JP2003519191A - Methods for obtaining thylakoids from photosynthetic organisms, plant fractions obtained therefrom, pure thylakoids, and methods of using thylakoids as ROS scavengers, photoprotectors, biosensors, biofilters and bioreactors - Google Patents

Abstract

This invention relates to a process by which an extract comprising integral thylakoids is obtained. The resulting extract is a potent dynamic antioxidant useful as a ROS (reactive oxygen species) scavenger. This extract is intended to be used for the treatment or prevention of diseases involving the generation of ROS, such as inflammatory diseases or cancer. This extract also finds a use as a solar screen because of its capacity to capture UV radiations and to dissipate the solar energy into heat.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the separation and recovery of thylakoids that exist in a substantially complete and natural state and are at least partially functional or activatable. The present invention also relates to the acquisition of other soluble and insoluble plant fractions obtained during the separation of thylakoids. Furthermore, the present invention provides a ROS scavenger, a photoprotectant, especially U.
It also relates to the use of thylakoids as photoprotectors against V rays and as biosensors, biofilters or bioreactors.

[0002]

[Prior art]

Antioxidants have become increasingly used in the biomedical field, so to speak, because they can prevent the formation and adverse effects of ROS (reactive oxygen species).

Plants and other photosynthetic organisms are particularly well adapted to resist the action of ROS, and in particular protect the essential organelle photosynthetic membrane called thylakoids from the oxidative damage and harmful effects of UV light. .

[0004] Sunlight plays a much larger role than we expected in sustaining our lives. That is, all food we eat and all fossil fuels we use are the products of photosynthesis, the process of converting the energy in sunlight into chemical forms of energy that biological systems can use. Photosynthesis is carried out by many different organisms, ranging from plants to bacteria. The best known form of photosynthesis is found in higher plants and algae,
And cyanobacteria, which are responsible for the majority of photosynthesis in the ocean, and their relatives. All of these organisms convert their gas to organic matter by reducing CO ₂ (carbon dioxide) to carbohydrates in a series of fairly complex reactions. The reducing electrons ultimately come from water, which is then converted to oxygen and protons. The energy for this process comes from the light absorbed by the pigments (mainly chlorophyll and carotenoids). Chlorophyll absorbs blue and red light and carotenoids absorb blue-green light, while green and yellow light is not effectively absorbed by photosynthetic pigments in plants. Therefore, light of these colors is reflected either on the leaves or on the pathways through the leaves.

Other photosynthetic organisms such as cyanobacteria (formerly known as cyanobacteria), red algae, are phycobilins that absorb visible light of a red or blue color that is not effectively absorbed by chlorophyll and carotenoids. It also has an additional dye, referred to as. Still other organisms, which incidentally appear to be fairly brown under many growth conditions, such as purple and green bacteria, contain bacteriochlorophyll, which absorbs in the infrared, in addition to the blue portion of the spectrum. These bacteria do not generate oxygen, but photosynthesize under anaerobic (anoxic) conditions. These bacteria effectively use infrared light for photosynthesis. Infrared light has a wavelength of over 700 nm and is invisible to the human eye. Some bacterial species can also use infrared light with wavelengths up to 1000 nm. However, ultraviolet light (<4
Most dyes are not very effective at absorbing (00 nm). Light with wavelengths below 330 nm becomes increasingly damaging to cells, but substantially all such short wavelength light is filtered out by the atmosphere (most notably the ozone layer) before reaching the ground. . Although most plants are capable of producing compounds that absorb UV light, increased exposure to light at about 300 nm has a deleterious effect on plant productivity.

Photosynthetic pigments are extremely diverse. There are many different types of (bacterio) chlorophylls, carotenoids and phycobilins that differ from each other in exact chemical structure. Dyes are generally associated with proteins that bring the dye molecules into proper orientation and alignment with each other. Light energy is absorbed by the individual dyes, but is not immediately used by these dyes for energy conversion. Instead, light energy is passed to chlorophyll in a special protein environment, where the actual energy conversion phenomenon takes place. That is, light energy is used to transfer electrons to adjacent dyes. The dye and protein that are involved in this actual initial electron transfer phenomenon are called reaction centers. A large number of dye molecules (100 to 50)
00) "collects" light, moving light energy to the same reaction center. The purpose is to maintain a high electron transfer rate in the reaction center, even at low light intensities. The name P680 has been assigned to the chlorophyll dye of the reaction center PSII, because the chlorophyll pair having its composition mainly absorbs light having a wavelength of 680 nm.

[0007]   Many antenna dyes transfer light energy to other antenna dyes, and to other antenna dyes.
By moving to a dye such as an antenna, its energy is trapped in a single reaction center.
Transfer that energy to the reaction center until it is "trapped". Overall movement process
Each stage of this energy transfer is not very efficient in order to avoid a large loss of
The association of various dyes with proteins must bring the appropriate dyes closer to each other,
In addition, the transduction efficiency is high by making the geometrical arrangement of dye molecules appropriate to each other.
I guarantee that. The exception to the law of protein-binding dyes is
It is a green bacterium with a tena system. A large part of these antenna systems
Up to thousands of bacteriochlorophytes that are used and not in direct contact with proteins
It is made up of "bags" (named chlorosomes) of the molecule. Chloroff
Is a link between excitation energy transfer and electron transfer in all photosynthetic organisms.
Used accordingly. The speed at which these transfer and transduction reactions need to occur is particularly critical.
It is important. After absorption of the quantum, the lifetime of the excited state is several nanoseconds (1 nanosecond (ns) is 10 ^-9 energy transfer and charge separation in the reaction center.
Must happen within this time. Very fast energy transfer rate between dyes
And the charge separation in the reaction center is 3 to 30 picoseconds (1 picosecond (ps)).
Is 10^-12s). The subsequent electron transfer phase is significantly slower (2
00 ps to 20 ms), nevertheless, the electron transport system is
It has a rate sufficient to allow at least a significant portion of the light to be used for photosynthesis. Chiraco
The dye should be retained if it is attempted to maintain its function during separation and purification.
It has a special organizational structure.

In many systems, the size of the photosynthetic antenna is flexible, and photosynthetic organisms that grow in weak light (eg, in the shade) have more antenna dye per reaction center than those that grow in higher light intensity. It seems that there are many in general. However, at high light intensities (eg, full day), the amount of light absorbed by plants exceeds the ability of electron transfer initiated by reaction centers. Plants have developed means to convert some of the absorbed light energy into heat, rather than necessarily using the absorbed light for photosynthesis. However, especially the first phase of photosynthetic electron transfer in plants is quite sensitive to excessively high electron transfer rates, and if the light intensity is too high, part of the photosynthetic electron transfer system may be blocked. This phenomenon is known as light inhibition.

The initial electron transfer (charge separation) in the photosynthetic reaction center initiates a long series of redox (reduction-oxidation) reactions, transferring electrons along a chain of cofactors, like a chain of bucket relays, and chlorophyll. Fill the above “electron holes”. All oxygen-producing photosynthetic organisms have two reaction centers, called photosystem II and photosystem I (abbreviated PSII and PSI), both of which are arranged in a special membrane called thylakoid. It is a dye / protein complex. In eukaryotes (plants and algae), these thylakoids are located in chloroplasts (organelles in plant cells) and are often found as stacked membranes (grass). Prokaryotes (bacteria) do not have chloroplasts or other organelles, and photosynthetic pigment-protein complexes are either found in the periplasmic membrane or in their invaginations (as found, for example, in purple bacteria). It is either in or within the thylakoid membranes that form much more complex structures within the cell (as in most cyanobacteria).

All chlorophylls in oxygen producing organisms are located in thylakoids, PSII, PS
I, or an antenna protein that supplies energy to these photosystems. PSII is a complex that causes water cleavage and oxygen evolution. Upon oxidation of the reaction center chlorophyll in PSII, an electron is withdrawn from a nearby amino acid (tyrosine) that forms part of the peripheral protein, which in turn obtains an electron from the water-cleavable complex. From PSII reaction center, electrons flow to free electron carrying molecules in the thylakoid membranes (plastoquinone), from which other membrane - flowing protein complex, the cytochrome b ₆ f complex. The other photosystem, PSI, also catalyzes photoinduced charge separation in a manner essentially similar to PSII. That is, the light is collected by the antenna and the light energy is passed to the reaction center chlorophyll, where photoinduced charge separation is initiated. However, in PSI the electrons are ultimately transferred to NADP (nicotinamide adenosine dinucleotide phosphate), whose reduced form can be used for carbon fixation. Reaction center chlorophyll is oxidized receives another electronic ultimately from cytochrome b ₆ f complex. Therefore, electron transfer through PSII and PSI results in the oxidation of water (which produces oxygen) and the reduction of NADP, and the energy for this process is light (two quanta for each electron transported through the entire chain). ).

The flow of electrons from water to NADP requires light and is coupled with the generation of a proton gradient through the thylakoid membrane. This proton gradient is used for the synthesis of the energetic molecule ATP (adenosine triphosphate). Reduced NADP resulting from ATP and photoreaction is used for CO ₂ fixation in a process independent of light. CO ₂ fixation requires several reactions called the Calvin-Benson cycle. The initial CO ₂ fixation reaction requires the enzyme ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO), which is either oxygen (which causes a process called photorespiration and does not cause carbon fixation), Or it can react with either CO ₂ . RuB
The probability that isCO reacts with oxygen with respect to CO ₂ depends on the relative concentrations of the two compounds at the reaction site. CO ₂ is by far the preferred substrate in all organisms, but photorespiration occurs at significant levels when CO ₂ concentrations are much lower than oxygen concentrations. In some plants (called C4 plants), some cells in the leaves (named vascular sheath cells) are mainly CO to increase local CO ₂ concentration and to minimize oxygen tension. ₂
It is involved in fixation and other cells (named mesophyll cells) are specialized in the photoreaction. ATP in mesophyll cells, CO ₂ and reduced NADP is used for (such as malate) 4 Synthesis of carbon organic acids, organic acid is transported to bundle sheath cells. Here the organic acids are converted to release CO ₂ and reduced NADP, which are used for carbon fixation. The generated tricarbonic acid is returned to mesophyll cells. Vascular sheath cells generally do not have PSII activity to minimize local oxygen levels. However, those cells retain PSI, presumably to support ATP synthesis.

The tissue of thylakoids is very sophisticated in extracting energy from light and transferring this energy to the proper location and / or dissipating it.
Its ability to dissociate charge and regenerate an electrically neutral state to reinitiate a change in charge allows its transfer and becomes effective (Blankenship et al., 1998).

Electron transfer between the five major components is very fast. Electron transfer from activated P680 to pheophytin takes less than 1 picosecond. Electron transfer ceases when all dyes return to a neutral charge and prepare for a new cycle.

Finally, the electron is transferred to a coupling factor to reduce NADPH required for ATP synthesis, which is later utilized for sugar synthesis.

The term “thylakoid” is used below and is meant to include organized photosynthetic membrane components obtained from a photosynthetic organism, whether eukaryotic or prokaryotic. When an organism has chloroplasts, thylakoids contain the following membrane components: PSII, cytochromes b ₆ and f, PSI and cofactors. When testing the integrity and functionality of thylakoids from plant material, it was measured between two reference points, the proximal side of PSII and the distal side of the coupling factor. For certain applications, thylakoids need not be active, although apparently intact. Such thylakoids are functional and at least as stable as any other antioxidant. Thus, by "active thylakoid" is meant a thylakoid that has the ability to activate when hydrated, rather than a complete but actively or passively inactivated thylakoid. In this case, the reaction center is inactive even though the thylakoid structure is substantially retained. Thus, an "inactive" thylakoid is a suitable antioxidant, although it does not have the same vitality as the active / activated form and does not have the same ability to regenerate or respond to ROS.

[0016]   Photosynthesis involves two basic processes that can be summarized in the following two reactions.

[Chemical 1]

During the first reaction in the presence of light, protons are removed from the chloroplast water to produce ATP. The second reaction consists in using NADPH and ATP in a series of reactions leading to the reduction of carbonic anhydride to sugars, mainly starch. These two reactions occur simultaneously and the product formed by step (1) is passed into the reaction of step (2).
Photosynthesis as a whole produces energy in the form of sugars and ATP molecules in the form of starch and sucrose.

[Chemical 2]

Photoactivation follows a route in thylakoids. Light is first collected by a light antenna (LHCII), its energy is passed to the reaction center (PSII) and finally to PSI, which also has an independent light collector (LHCI). The thylakoid has a function of moving light energy to an appropriate place in order to collect light and promote photosynthesis. ATP and sugar synthesis does not occur in thylakoids but in other compartments of the chloroplast.

Chlorophyll is the main active dye. Carotenoids have multiple roles depending on where they are located. The first role is as a light-harvesting agent, causing energy transfer from carotenoids to chlorophyll. The second role is as a photoprotectant, where energy transfer occurs in opposite directions between chlorophyll and carotenoid. The carotenoid singlet state has more energy than singlet chlorophyll, while the carotenoid triplet state has less energy than triplet chlorophyll. Singlet carotenoids mainly act as light-harvesting agents, passing light energy to the singlet chlorophyll molecule, while triplet chlorophyll has its energy, because the energy state has a natural tendency to move from high energy levels to low energy levels. Would readily transfer to the triplet carotenoid, whereupon the latter acts as a photoprotectant in the reaction center. Carotenoids assume different conformations when associated with an antenna or reaction center, but are believed to be responsible for the energy state upon activation of that conformation. The "cis" configuration is associated with photoprotection at the reaction center. The configuration of "all transformers" is related to the function of the antenna light collector.

Energy transfer is only effective under conditions where the dyes are in close proximity to each other and in certain tissues. Therefore, if it is desired to purify thylakoids that are either active or fully activating, it is very important to keep the membrane intact without disturbing the natural organization of the dye.

One advantage of recovering intact thylakoids is found in their ability to handle ROS.
Such ROS are intended to include free radicals (including superoxide) as well as non-radical oxidants such as singlet oxygen ( ¹ O ₂ ) and peroxides. A good review of the definitions and origins of these species can be found in WO 94/133.
Found in No. 00. The contents of all references cited above and below are incorporated herein by reference.

Free radicals are atoms, ions or molecules containing unpaired electrons. Free radicals are usually unstable and show a short half-life. Elemental oxygen is highly electronegative and readily undergoes one-electron transfer from cytochromes and other reduced cellular components. Therefore, some of the O2 cells to the aerobic respiration consumes, superoxide radical ^(· O ₂ ^-) receives a monovalent reducible to. Subsequent ^· O ₂ ^- by monovalent soluble reducing the hydrogen peroxide (H ₂ O _2), hydroxyl radical ^(· OH) and water is produced.

Free radicals are cytochromes P-45 for aerobic respiration, drugs and xenobiotics (eg trichloromethyl radical, ie CCl ₃ ^· formed by the oxidation of carbon tetrachloride).
It can occur from many sources, including zero-catalyst monoatomic oxygenation reactions, and ionizing radiation. For example, when tissues are exposed to gamma rays, most of the energy stored inside the cells is absorbed by water, causing the breaking of oxygen-hydrogen covalent bonds in water,
1 electronic, results leave one electron also on oxygen on hydrogen, two radicals H ^· and ^· OH
Create. The hydroxyl radical ^, OH, is the most reactive radical known in chemistry. It can react with biomolecules, initiate chain reactions and interact with nucleic acid purine or pyrimidine bases. In fact, radiation-induced carcinogenesis can be initiated by free radical damage. In addition, for example, the “oxidative burst” of activated neutrophils produces abundant superoxide radicals, which is considered to be an essential factor in producing the cytotoxic effect of activated neutrophils. Reperfusion of ischemic tissue also produces high concentrations of oxygen radicals, usually superoxide. Furthermore, physiological regulators in a reaction with peroxynitrite ions ONOO the nitric oxide ^- by endothelial cells that form, although superoxide may physiologically generated hydroxyl radicals salt thereof disintegrates ^• May produce OH. Other sources of oxygen radicals are electron “leakage” from destroyed mitochondrial and endoplasmic reticulum electron transport systems, prostaglandin synthesis, catecholamine oxidation and platelet activation. Many free radical reactions are highly toxic to cellular components. These reactions crosslink proteins, mutate DNA, and peroxidize lipids. Once formed, the free radicals interact to give rise to other free radicals and non-radical oxidants such as singlet oxygen ( ¹ O ₂ ) and peroxides.

Singlet oxygen is a particularly harmful compound involved in the initiation or persistence of many diseases and disorders. Singlet oxygen is also involved in the degradation of proteins such as chlorophyll. This is why the photoprotection afforded by the presence of carotenoids is important. Carotenoids protect the life and activity of chlorophyll and also protect the integrity of the membrane by preventing protein denaturation. Carotenoids can trap the energy of triplet chlorophyll molecules. as a result,
It becomes a triplet carotenoid molecule, which self-regenerates while dissipating heat, thereby avoiding the accumulation of triplet chlorophyll molecules and minimizing the probability of degrading chlorophyll.

However, in the presence of excess light, injury can occur, which may result from the formation of “triplet state” chlorophyll. In the triplet state, the two electrons in the outer shell have the same spin orientation, not the opposite. This triplet chlorophyll readily reacts with oxygen, resulting in the production of highly reactive singlet oxygen, which can damage proteins. To combat this damaging response, carotenoids are usually present in close proximity to chlorophyll. Many carotenoids effectively "quench" the triplet state of chlorophyll, thereby avoiding the formation of singlet oxygen. The free form of chlorophyll is highly toxic in the presence of oxygen in the light, because in such circumstances a close interaction with carotenoids may not be realized. Therefore, all the intracellular chlorophylls in aerobic organisms are bound to proteins, and in general carotenoids are bound to the same proteins.

The main difficulty in measuring enzyme kinetics on a fairly short time scale (less than 1 ms) is that “traditional” enzymatic reactions require a mixture of substrate and enzyme, which is usually a fairly long time. Is to take. Kinetic analysis of light-driven reactions such as photosynthetic electron transfer has a great advantage in this respect. This is because only a light pulse that can be much shorter than 1 ps can trigger the reaction. Moreover, many of the components involved in electron transfer have different absorption spectra depending on whether they are in the oxidized or reduced form. Using laser spectroscopy or more standard optical spectroscopy, it is fairly easy to track the surrounding electrons on a time scale of between 1 ps and a few ms. The initial charge separation occurs at a few ps and the reaction becomes progressively slower as components further away from the reaction center participate in the reaction. Electrons and "electron holes" are physically fast due to long distances (generally, electrons move about 2 nm to reach the opposite side of the membrane within 1 ns after charge separation) due to the fast reaction rate. , So that the reverse reaction (charge binding) is no longer favorable. During the redox reaction, which often involves one-electron transfer, the unpaired electrons on the reactants formed transiently are detected by electron paramagnetic resonance (
EPR) and inductive techniques (including ENDOR, electron nuclear double resonance and ESEEM, electron spin echo envelope modulation). Many of these techniques can be used to track the rate of redox reactions, providing detailed information about the electron spin distribution, etc. Therefore, photosynthetic membranes and reaction centers occupy an important position as experimental systems in biochemistry and biophysics.

[0027]   The antioxidant properties of compounds such as chlorophyll are exemplified in formula (1).

[Chemical 3]

Chlorophyll excited in the presence of oxygen is in a triplet state ( ³ Chl ^* ) and is inactivated to return to the ground state by generating singlet oxygen (toxic species) in the cell. . Plants have found effective means by which the problem of singlet oxygen overproduction can be overcome. Plants transfer the energy of chlorophyll to pigments with lower energy states. Its pigment, a carotenoid (Formula 2), is abundant in plants.

[Chemical 4]

Triplet chlorophyll has more energy than the corresponding carotenoid, but the opposite is true for the singlet state. As shown in equations 3a and 3b, the activated singlet carotenoid transfers its energy to the chlorophyll molecule, resulting in the chlorophyll molecule being activated in the singlet state.

[Chemical 5]

Triplet state carotenoids are inactivated without forming harmful oxygen species. Equation 4 shows that carotenoids are inactivated by returning to the ground state and dissipating heat.

[Chemical 6]

In order to preserve the properties of the pigments in the extract, it seems important not to generate ROS, but it is also important to remove such ROS which may occur during the separation. To achieve this, we favored a method of reversing the equilibrium in Eq. As a result, the inverse of Equation 1 is found in Equation 5.

[Chemical 7]

In order to avoid the retrograde of Equation 5, the activated triplet chlorophyll molecule must be in close contact with the ground state carotenoid, which receives the energy transferred and Dissipates as heat. Thus, the reversibility of Equation 5 is limited as long as the chlorophyll and carotenoid dyes can be found in close proximity to transfer energy to each other.

From equation 5 above, it is clear that it is preferable to take every conceivable means of maintaining both pigments (chlorophyll and carotenoids) in the ground state in order to obtain an extract with optimal activity. Is. An isolated carotenoid, such as a carotenoid that is not organized within the thylakoid structure, would not be able to effectively quench the triplet chlorophyll molecule. The advantage of having organized pigments is that the extract retains the vitality of the native thylakoid membranes so that they can capture ROS, transfer energy, and reinitiate a new activation cycle. It seems that you will acquire the ability to return to the state. This vitality and ability to regenerate is typical of organized pigments. It should be emphasized that the above reaction occurs spontaneously even in the absence of light. This observation is important from a therapeutic point of view, as the in vivo administration of thylakoid extracts appears to rule out the presence of light.

Thylakoids with optimized configuration and carotenoid ratio appear to retain sufficient activity, especially against ROS. Such antioxidants may be useful in reducing the expression of diseases and disorders involving ROS production. Such diseases and disorders may have etiologies associated with inflammation, cancer and radiation exposure. Such diseases and disorders include skin such as burns, sunburn, psoriasis, dermatitis; trauma, stroke,
Brains such as Parkinson's disease, neurotoxin, dementia, Alzheimer's disease, joints such as rheumatoid arthritis, gastrointestinal tract such as diabetes, pancreatitis, endotoxin liver injury, intestinal ischemia, etc .; Vessels such as atherosclerosis; red blood cells such as Fanconi anemia and malaria; heart such as coronary artery thrombosis; lungs such as asthma and COPD; kidneys such as transplantation and glomerulonephritis; inflammation, cancer, ischemia-re Perfusion status, drug poisoning, iron overdose, malnutrition,
Includes those that affect multiple organs such as alcoholism, radiation, aging, amyloid disease. Literature on the involvement of ROS in some diseases is:

[0035] Skin:       Burn Youn, 1992       Tans Golan, 1994       Psoriasis Lange, 1998 a, b       Dermatitis Polla, 1992 brain:       Trauma Juurlink, 1998       Stroke El Kossi, 2000       Parkinson's disease Ebadi, 1996       Neurotoxin Foler, 2000       Alzheimer's disease Smith, 2000 joint:       Rheumatoid arthritis Cimen, 2000 Gastrointestinal tract:       Diabetes Gerber, 2000       Pancreatitis Sakorafas, 2000       Endotoxin liver injury McGuire, 1966       Intestinal ischemia Lai, 2000 eye:       Cataract development Eaton, 1994       Retinopathy of Prematurity Hardy, 2000       Degenerative retinopathy Castagne, 2000 Vessel:       Atherosclerosis Singh, 1997 Red blood cells:       Anemia Anastassopoulou, 2000       Malaria Ginsburg, 1999 heart:       Coronary thrombosis Chen, 1995 lung:       Asthma Montuschi, 1999       COPD Montuschi, 2000 kidney:       Glomerulonephritis Barros, 2001 Multi-organ:       Transplant Jonas, 2000       Inflammation El-Kadi, 2000       Cancer Prior, 2000       Ischemia Lewen, 2000       Drug Addiction Sinha, 1990       Iron overdose Karbownik, 2000       Malnutrition Olszewski, 1993       Alcoholism Lieber, 1997       Aging Cadenas, 2000       Radiation Bednarska, 2000       Amyloid disease Floyd, 1999

In addition to therapeutic applications, it has been found that the thylakoids of the invention can advantageously replace chloroplast-derived compositions in the art that have been tested as biosensors or biofilters or bioreactors. Although the art teaches these particular uses, these chloroplast-derived compositions lack stability and degrade very rapidly, making these uses impractical from a commercial perspective. . Thus, stable and vigorous thylakoid extracts could advantageously replace these non-functional chloroplast derived compositions.

Biosensor: The detection of toxic products is important for assessing the environmental risk associated with the presence of pollutants. Effective organisms will normally use organisms and will meet the following minimum requirements: i) they should correspond to the natural environment, ii) they should be reproducible, and iii) they should be reliable so as to give no or little false result. And iv) they should be sensitive.

Toxicity detection should also provide sufficient flexibility to analyze various contaminated samples. Ideally, toxicity should be investigated and sensed immediately. Toxicity detection has applications in at least three sectors of industry, the paper industry, contaminated soil analysis and agriculture. In all of the above cases, information on the presence of pollutants is needed to quickly correct the undesirable situation.

The main problems faced by practical technology for sensing toxic products are trout and Daphne
With organisms such as a magna, there is a long delay of 48 to 96 hours that occurs in obtaining the results of biological tests. A good detection device appears to be a device that differs from the traditional biological tests available by using a material that allows the immediate and continuous measurement of wastewater contamination. There are several biodetectors on the market that measure the fluorescence generated by the photosynthetic activity of plants, but it would be ideal if there were a system that could measure the charge induced by the presence of light and modulated by the presence of contaminants. I think that the. This technique seems to be much cheaper than the fluorometric technique. If there is a technique for evaluating photosynthetic activity on the entire thylakoid material, a specific protein complex,
That is, it seems to be preferable to the fluorometric method for measuring the activity of PSII. Therefore, devices containing thylakoid materials would have the advantage of measuring toxicity with a broader spectrum of action. Such a detector would measure the number of electrons produced at a given light intensity. The current obtained after a few seconds (Ep _max ) should be proportional to the photosynthetic activity of thylakoids. If plotted photocurrent versus the concentration of the contaminant, typical S-shaped curve is obtained, it should estimate the EC ₅₀ of determined thereon.

Photocurrent was already measured in 1976 by Allen and Crane. Electron transfer has been found to be a reliable and representative measure of overall photosynthetic activity and physiological health of plants. From the work of Allen and Crane, it appears that extracts with great stability, together with the vitality and ability to regenerate pollutants and reaction conditions to light, are highly preferred over known devices. The detector will measure the number of electrons produced at a given light intensity. Maximum photocurrent obtained after 2-3 seconds (Ip _max )
Should be proportional to the photosynthetic activity of the thylakoid membrane. A typical sigmoidal curve can be obtained by plotting Ip _max against the concentration of photosynthetic inhibitors (pollutants or pollutants) present in the photoelectric conversion chamber. Its inhibitor potency can be easily assessed (IC ₅₀ ).

It is assumed that the detection device comprises a white light source, a photoelectric conversion chamber containing two electrodes, a detection means for measuring the photoinduced current and a computer means for collecting and processing the data (current). Be done. A liquid sample containing the toxic, pollutant or pollutant to be identified or measured is contacted with the thylakoid membrane extract. After introducing the mixture into the chamber, irradiation is carried out for a short time (less than 1 minute). The device or instrument would process and analyze multiple samples simultaneously.

Biofilter / Bioreactor: Since the photosynthetic device in a plant can capture and accumulate molecules having an affinity for each component thereof, the extract of the present invention has the same ability as the plant itself. Imagine having. Moreover, it is believed that the extract of the present invention acts as a bioreactor since it can process some of the capture molecules. Captured easily molecule, for example, herbicides, insecticides, fungicides, urea, ions and heavy metals, as well as _{O 3, CO, H 2 S} , NO, CO 2, gas such as O _2. The biofilter of the present invention is versatile and appears to be resistant to temperature changes.

There is no practical process that teaches how to recover intact functional thylakoids that can retain activity for a practical amount of time.

It is clear from the above that plants have excellent natural ability to cope with threatening situations. The thylakoids are particularly adapted to resist and adapt to these extremes.

US Pat. No. 4,698,360 describes plant extracts containing proanthocyanidins useful as free radical scavengers. The method of making this extract comprises the following steps. a) Obtaining crude powder of cabbage skin, b) Extraction in boiling water, c) Separation of liquid from solid, d) Cooling of liquid to ambient temperature, e) Filtration, f) "Salting out". Removal of waste by precipitation, g) extraction of the active ingredient into ethyl acetate, h) drying of the organic phase, i) resuspension of the solid and reprecipitation of the active ingredient with chloroform, and j) resuspension of the solid. And then advanced purification.

This reference relates to the isolation of a particular type of active ingredient, in other words not to the preparation of thylakoids which are believed to contain the major part of the photosynthetic component in which the dyes are not separated from one another.

This reference is, in fact, representative of the general teachings in the general art to which the invention pertains. This prior art relates systematically to the isolation of one or more given plant constituents and contains an intact thylakoid containing a major portion of the constituents preserved in a complete and functional state. It's not about separation.

In Planta 164: 487-494, Glick et al. (1985) describe the variation in stoichiometry of photosystems II and I (PSII / PSI) when exposed to various types of light peas. is doing. The electron transfer capacity of PSI and PSII has been measured in the presence of indicators such as 2,5-dimethyl-p-benzoquinone and NADP, which are specific indicators for PSII and PSI, respectively. A non-activating light environment, green light, is used, but it is not used to condition plants in a process that aims to separate intact activatable thylakoids. . The reference is essentially concerned with the study of chloroplast composition, not retention of thylakoid activity as a function of given light quality and intensity. Rather, plants are conditioned in a variety of lights with red wavelengths depleted or enriched. This reference does not relate to the fact that the photosynthetic dyes should be in close proximity to each other to promote energy transfer between chlorophyll and carotenoid and to facilitate free radical scavenging. Therefore, the conditions that would result in isolation of the photosynthetic dye in the thylakoid in its natural state are not specifically taught or mentioned in this reference.

Mason et al. (1991) teaches a method for separating chloroplasts in Plant Physiol. 97: 1576-1580, which method does not use a homogenization dispersion step. A step of forcing the plant suspension through a No. 27 needle at a flow rate of 0.5 mL per second is utilized. The plant solution contains a buffer containing 0.3 M sorbitol at pH 7.5. The preparation that has been forced through the needle is centrifuged in a Percoll gradient to separate chloroplasts from other components, including thylakoids. Therefore, this process differs from the process of the present invention, which is essentially aimed at the recovery of thylakoids using quite simple steps and reactants and is easy to scale up. No light conditions are mentioned in this reference. Furthermore, it is clear that the conditions that keep chloroplasts intact are not the same as the conditions that degrade chloroplasts. In the method of the present invention, the chloroplasts are degraded, but the thylakoid membrane is recovered substantially intact. Therefore, this reference cannot teach the present invention.

Canadian Patent Application No. 2110038 describes a method of stabilizing plant extracts. However, these extracts are cell fluids and juices, not thylakoid membranes. This reference does not mention the removal of water, the natural electron donor, from the film to stabilize the thylakoids.

In view of the above, no practical method is taught in the art to lead to the separation of intact functional thylakoid membranes. Furthermore, the conditions for stabilizing the thylakoid component are not taught. Finally, the use of discrete thylakoid membranes to remove cellular components from ROS is also not taught.

Thus, a method of obtaining active thylakoids that can maintain their integrity and can be activated for an optionally tolerable time and, upon reactivation, can act as antioxidants due to their ROS scavenger activity. The development of is an unsolved challenge. Despite the increasing availability of literature on photosystem components, no practical method has been published that teaches conditions for separation and storage of thylakoid activity.

Moreover, scavenging free radicals is expected to protect other plant components, as free radicals can cause the degradation of many cellular components. Thus, the method of the invention appears to produce plant components other than thylakoids in improved yields.

There is a need for strong antioxidants, especially in the pharmaceutical field, so a method of providing any such antioxidant, as well as the value of the antioxidant itself, not only with sufficient potency, but also with sustained activity. Will be greatly appreciated. Furthermore, there is a need for sensors or detectors, capture materials or filters, bioreactors or biomolecule production devices.

[0055]

[Problems to be Solved by the Invention]

The present invention aims to provide a simple method for obtaining an extract with functional thylakoids. The invention also provides methods for purifying thylakoids from other cellular components. It is another object of the present invention to provide a stabilized extract containing unseparated or separated thylakoids. The stabilized extract is essentially free of electron donors that appear to activate thylakoids. Since the most abundant electron donor is water, the stabilized extract is therefore preferably anhydrous. Water can be driven off, for example, by solvent or drying. As a result of trying an amphoteric solvent, especially a surfactant such as propylene glycol, the result was successful. This type of solvent has the advantage that it does not decompose the structural components of the membrane, it replaces water molecules and prevents the formation of aggregates when redissolved in aqueous solution. The stabilized extract has a longer shelf life without substantial loss of activity unless an electron donor such as water is added. The stabilized extract is reconstituted unprepared before use to initiate activation. The activity of the extract once activated is much longer lasting than any other known antioxidant, indicating a level of regeneration of activity rather than immediate exhaustion. Furthermore, the efficacy of antioxidants adapts, ie increases or decreases, depending on the extent of oxidative damage.

[0056]

[Means for Solving the Problems]

  According to the invention, a method as specified in claim 1 is provided.

The present invention is a method of obtaining an extract obtained from a photosynthetic organism containing thylakoids, comprising the steps of: a) providing a suspension of biological components containing thylakoids, and b) between 1 and 1.3. In a medium having a viscosity of 2 and a pH higher than 2 and lower than 10 while destroying its biogenic constituents while maintaining the thylakoid intact, the medium being added in a volume calculated according to the formula: ((Volume of medium + water content of plant components) / dry weight of plant components)> 10, thereby obtaining a first extract consisting essentially of thylakoids, cell debris / membrane and liquid phase, said thylakoids Provides a step wherein the comprises a complete photosynthetic dye.

[0058]

DETAILED DESCRIPTION OF THE INVENTION

  The calculated value of the above formula is preferably comprised between 25 and 150.

The pH of the medium is preferably comprised between 5 and 8, more preferably between 7 and 7.5.

The suspension of step a) is obtained by mechanically dispersing the biological component or tissue in the medium.

In a preferred embodiment, prior to step a) the organism is subjected to conditioning parameters selected from light, osmotic pressure, heat, coldness, freezing, drying, hormones, chemical and biological inducers. Perform the step.

In a most preferred embodiment, step a) is preceded by the step of conditioning said living body in a light environment with a wavelength comprised between about 500 and 600 nm and step b) under the same light environment. .

The viscosity is achieved in part by adding sugar. Sugar may be added at concentrations as high as 1.5M or higher. Preferably, sugar is added to achieve a sucrose concentration of about 0.2 to 0.4 M in the solution, or a viscosity equivalent to 0.2 to 0.4 M sucrose.

Particular examples of media used in the method are Tris or acetate or ascorbate buffer (20 mM, pH 7.0-7.5) and sorbitol or sucrose 3
It is 50 mM.

The process according to the invention is further essentially composed of the following steps c), namely separating thylakoids, cell debris / membrane and liquid phase from each other, respectively in separated thylakoids, cell debris and membranes and liquid phase. May comprise the steps of forming second, third and fourth extracts that are composed of:

The separation step was carried out in particular on the basis of the differences in the sedimentation coefficients of thylakoids, cell debris and membranes and of the liquid phase.

A particular example of such a separation step is that when centrifuging the first extract for 10 minutes at 10000 g in a tube with a filter on top of the tube, the filter is
Includes that the thylakoids and liquid phase pass through the filter while the cell debris and membranes have suitable porosity on which the thylakoids form a pellet at the bottom of the tube. Alternatively, crude purification can be achieved by first collecting cell debris and membranes, eg by squeezing and / or filtration, and then finer purification, eg separating thylakoids from the liquid phase.

After separation, each of the first to fourth extracts can be stabilized by adding the next step d). The step is to deactivate and stabilize the photosynthetic dye by eliminating any electron donors from each of the above extracts, preferably in the presence of sugar (which may protect the component from cold). is there. At this stage, the second and third extracts are of particular interest.

The first electron donor envisaged is water, so the extract is treated to be anhydrous.

After step c), the water may be removed by vacuum freeze-drying or by exchanging the water with a non-altering amphoteric solvent or a surfactant, in which case the non-altering refers to the thylakoid structure. It means that the component cannot be dissociated or damaged.

[0071]   An amphoteric solvent that has been used successfully has been propylene glycol.

It is a further object of the present invention to provide a product resulting from said method.
First, a pure thylakoid extract with the ability to be activated is provided. Stabilized extracts are preferred. The aforementioned third extract rich in thylakoids and cellulosic substances is also within the scope of the present invention, ie in stabilized form. The stabilized form of thylakoids may be dried or in a medium composed of amphoteric solvents such as propylene glycol. The former is an insoluble state or a suspension, and the latter is a solution. The thylakoid-containing extract is reactivated in the presence of an electron donor. The envisioned first electron donor is water. Once activated, the extract is ROS
Acts as the vital scavenger of.

For nutritional, cosmetic and pharmaceutical applications, this scavenger activity is
It is useful for the treatment and prevention of diseases and disorders mediated by the formation of erythrocytes, especially those having an etiology associated with inflammation, cancer or radiation exposure.

The first scavenging and protective effect is exploited for radiation in the UV spectrum. Thus, topical applications for thylakoids and topical compositions containing thylakoids are within the scope of the invention.

It has been found that thylakoids have the ability to form photon absorbing films or coatings on body surfaces such as skin and mucous membranes. This property appears to be independent of ROS scavenger activity. Therefore, the thylakoid-containing extract acts as a filter for radiation, i.e. radiation in the ultraviolet range, whether in an activatable form or not. When the extract further has a functional thylakoid, it plays a dual role as a UV filter and ROS scavenger. The extract is further used in any of the methods, compositions, devices for detecting or capturing a molecule, or producing or treating a molecule having an affinity for thylakoids, or interfering with its activity. Good.

Examples of such molecules are herbicides such as triazines (eg atrazine and diuron herbicides), quinones, chlorpromazine, urea, formaldehyde, alkylaminocyanoacrylates, trypsin, cyanoacrylates, tris, adenine. Derivatives, disulfirane (metal chelating agents), acetyl CoA carboxylase, digitonin, heavy metals (eg Cu, Zn, Cd, Pb, Hg ...), SO ₂
, NO ₂ , NH ₂ OH, CO ₂ , CO, O ₃ , O ₂ , H ₂ S, calcium antagonist (calmodulin type)
, Sulfate, sulfite, bisulfite, nitrite, acetate (salt), lactate (salt), NO ₃ ^{-,
_{HCO 3 -, HCO 2 -,}} F -, NO 2 -, HSO 3 - , and the like of the anion.

DESCRIPTION OF THE INVENTION The present invention is illustrated below with reference to specific embodiments and accompanying figures, whose purpose is to illustrate the present invention and not to limit its scope.

DESCRIPTION OF THE INVENTION The present invention is illustrated below with reference to specific embodiments and accompanying figures, whose purpose is to illustrate the present invention and not to limit its scope.

As used herein, an “antioxidant” is a substance that, when present in a mixture or structure containing an oxidizable biomatrix, significantly delays or prevents the oxidation of that biomatrix. Is. Antioxidants are biologically important reactive free radicals and other ROS (singlet oxygen, ^· O2-, H ₂ O ₂ , ^· OH, HOCl ferryl, peroxyl, peroxynitrite, alkoxyl ...). Can act by scavenging or preventing their formation, or by catalytically converting free radicals or other ROS into less reactive species.

The antioxidants of the invention, when added to cell culture media or assay reactions, compared to co-progressing cell culture media or assay reactions that have not been treated with antioxidants,
It is considered to be such if it causes a detectable decrease in the amount of free radicals such as superoxide and non-radical ROS such as hydrogen peroxide and singlet oxygen. Appropriate concentrations (ie, effective dose) can be determined by experimental dose-response curve generation, QSAR
It can be determined by a variety of methods, including the use of methods and prediction of potency and efficacy of homologous substances by molecular modeling, and other methods used in pharmaceutics.

To treat, prevent, or alleviate symptoms associated with ROS-related diseases and disorders,
Alternatively, the present invention is intended for use in the medical field to reduce the occurrence of such diseases and disorders. Such a disease or disorder refers to an individual's condition that results, at least in part, from the production or exposure to free radicals, particularly oxygen radicals, and other ROS in vivo. There is increasing literature and knowledge on the involvement of ROS in the etiology, even if there are only a few pathologies based on a single cause. For this reason, the term "ROS-related disease" may mean that ROS injury is responsible for the pathology of the condition, or that it is a free radical inhibitor (eg desferrioxamine), scavenger (eg tocopherol). , Glutathione) or a catalyst (eg, SOD, catalase) to reduce symptoms, increase survival, or provide other detectable clinical benefits in treating or preventing a medical condition. , Including conditions recognized in the art as conditions that are shown to produce a detectable benefit. By way of example, but not by way of limitation, conditions discussed herein, such as ischemic reperfusion injury, inflammatory disease, systemic lupus erythematosus, myocardial infarction, stroke, traumatic hemorrhage, spinal cord trauma,
Crohn's disease, autoimmune diseases (eg rheumatoid arthritis, diabetes), cataract formation, uveitis, emphysema, gastric ulcer, oxygen poisoning, neoplasms, unwanted cell apoptosis, radiation sickness are considered as ROS related diseases. Furthermore, many inflammatory diseases and disorders are known to benefit from the present invention as ROS are known to mediate inflammatory processes. For example, the "oxidative burst" of activated neutrophils is believed to produce large amounts of superoxide radicals, which is an essential factor in causing cytotoxicity of activated neutrophils. Furthermore, since neutrophils are involved in premature death of grafted or transplanted tissues and cells, antioxidants appear to increase the early survival of transplanted or grafted cells essential for successful transplantation.

[0082]

【Example】

The present invention relates to isolated thylakoids and methods of isolating thylakoids that will constitute potent antioxidant molecules with scavenger activity against ROS. This antioxidant is of natural origin and should not have any toxic or detrimental effects when used in a corresponding concentration. The antioxidant can also be stabilized, which ensures its stability over time and thus its reasonable shelf life. Stabilization is done by withdrawing an electron donor (ie, a water molecule), which keeps the thylakoid in a quiescent state. Thylakoids add an electron donor (ie hydration)
Is activated by.

Preparation or Conditioning The first step taken before entering the step for recovering the thylakoids in crude suspension may be a condition setting step. This condition setting is performed depending on the case, and the composition of the extract can be changed. In order to optimize the levels of pigments (ie chlorophyll and carotenoids) in the inactive state, the conditioning step is performed under the same conditions as the working conditions, eg under green light or in the dark. Under such circumstances, chlorophyll is preferably in the singlet state, while carotenoids are preferably in the basic state. When ready in this way, carotenoids are activated and ready to take in energy coming from the triplet chlorophyll (photoprotection).

Further protection of the thylakoid dye by adding another xanthophyll such as violaxanthin to the solution of extraction, or a narrow wavelength band (465-565).
It is also possible to increase the number of carotenoids by acting under light with (475 nm).

Furthermore, it is also possible to enrich the organism, ie the plant and its extract, for some specific constituents by subjecting the organism to certain conditioning steps other than light conditions. Such other condition settings include osmotic stress, heat, cold, freezing, drying, hormones, and chemical and biological inducers. All of these conditioning parameters lead to a response of the sensitive organism, which then leads to the enrichment of said certain components.

To give an example of this, heat treatment would promote the accumulation of heat shock proteins, which would be useful in treating diseases or disorders associated with ROS (ie, arthritis). The main purpose of the steps of the method is to maintain the integrity of some valuable components, namely the molecular components of thylakoids, and to control the state of the molecules, preferably to their basic functional state. Is.

Obtaining Crude Extract When starting with a whole organism or tissue, such as a plant tissue or whole plant,
The first step in this method is a dispersion step such as a homogenization step. The plant tissue is mechanically ground, for example. Mesophyll tissue (leaves or needles)
May be cut into small pieces using a rotating knife or a commercially available rotary cutter for recovery by a homogenizer. Any means leading to dissociation of the cellulosic material to expose the thylakoids would be suitable.

In addition to operating under a light source (green light, λ = 500-600 nm) that optimally minimizes the luminous flux, ideally it also aims to increase the cell density and prevent any enzymatic degradation. And the working conditions include a working temperature of about 2 to 20 ° C, preferably less than 4 ° C. Working conditions also include hypertonic conditions using hypertonic agents such as sugars. These conditions achieve optimum viscosity and flowability. Specific examples of homogenization buffers are as follows.

[Table 1]

The pH of the solution can be changed from 2 to 10 and preferably from 5 to 8, and more preferably can be maintained at a value near neutrality of 7 to 7.5 (FIG. 1).

When the reference plant is spinach, the ratio of leaf tissue wet weight (g) / buffer volume (mL) is about 1/3. Therefore, the above formulation is suitable for extracting thylakoids from 100 g of spinach. The plants are mixed with buffer, for example in a domestic blender, and homogenized for about 30 seconds. The plant supply can be varied like the volume of medium. The buffer itself is suitable to maintain a near neutral pH. For example, the Tris buffer above can be replaced with an acetate or ascorbate buffer. Both displacement buffers are acceptable for human consumption, and ascorbic acid has the advantage of supplying vitamin C to the consumer. Sorbitol was added to maintain the integrity of the membrane and ensure a viscosity of about 1 to 1.3 (Figs. 2 and 3), but other saccharose, fructose or other turbinado such as turbinado. 0.2 to 1. With any suitable sugar.
It can be replaced with a concentration that achieves the same effect as 5M (preferably 0.2-0.4M) sorbitol. 0.2-0.4M sucrose would be an acceptable and inexpensive ingredient (Figure 4). Buffer components such as MgCl ₂ , NaCl, ascorbate / acid are not considered necessary for this method, but they may help recover more activity or maintain activity for longer periods. Not (Fig. 5).

A pH near neutral was preferentially chosen to maintain optimal H ⁺ ion concentration. Sugar and pH are important parameters to prevent dissociation of photosynthetic pigments. The specific gravity of cell liquor is maximized when it is operated in a cold or cold environment, that is, at 4 ° C. or lower (see FIG. 6, where FRTS / 1 symbolizes this extract). The low temperature also protects the components from enzymatic degradation. All of these homogenization conditions release the membrane structure from the organic tissue structure within its chloroplast without substantially affecting the tissue structure of the thylakoid molecular structure. Thus chloroplasts are loosened without destroying or splitting thylakoids. The surface of any cellulosic deprotected cellular components is thus increased.

It was convenient in this method to use the plant tissue directly in the extraction solution. However, if it would be advantageous to use pure chloroplasts or preparations enriched in chloroplasts or even preparations of other photosynthetic organisms with or without chloroplasts, do so as well. It is possible. It is also clear that the cultured cells or tissues can replace the whole plant.

Starting from spinach leaves, the yield of thylakoids is significantly better according to the following formula: α / β> 10 where α is the wet weight / dry weight ratio and β is wet weight / (medium It is the ratio of volume + water content of plant components).

[0094]   Therefore, ((Volume of medium + water content of plant components) / dry weight of plant components)> 10 Becomes

[0095]   More precisely (preferably) ((Volume of medium + water content of plant components) / dry weight of plant components) = 25-150 Is.

It is worth mentioning that the yield may vary depending on the volume of buffer selected and the water content of the plant selected. For example, pine needles have a much less significant amount of endogenous water content than do spinach leaves. Taking into account all the parameters of the above formula for the isolation of thylakoids from pine needles, in order to obtain a uniform wet weight of plant material, the volume of buffer should be increased compared to spinach leaves. I have to.

The crude extract obtained alone constitutes the first fraction which can be used as such, or after dehydration or further fractionation.

Separation of Plant Fractions A homogenization step is followed by a separation step. Thylakoids are separated from cell debris and soluble components based on their difference in sedimentation coefficient.
The thylakoid sedimentation coefficient is higher than that of cellular organelles. The thylakoids were centrifuged for 10 minutes at 10,000 xg in a handheld bucket. 10,000
Centrifugal forces of less than xg but more than 3,000g can be used with the centrifugation time adjusted accordingly (Figure 7). Optimal affordability for thylakoid pellets was obtained at 10,000-12,000 xg for 10 minutes. It is possible to adapt any other speed and time for equivalent results. Large scale processing is intended for different speeds and times. During settling, the thylakoids pass a filter corresponding to: 0.002 ≦ X ≦ 0.2 where X is calculated by integrating the openings per wire diameter (all millimeters). Cell debris and membranes are stopped by this filter in the upper part of the centrifuge tube. Thus, the thylakoid-containing bottom pellets are easily recovered and the pellets may be used immediately, or may be further fractionated, or sometime stabilized for future use. Of course, some other method may be used to achieve the same purpose of isolating thylakoids. For example, it is possible to use a density gradient such as a sucrose gradient. Any chromatographic or affinity solution and method may be used. According to the above-identified method, it is believed that large scale processing does not achieve overall and high purity separation in one step. Therefore, the overall purification will be done first in the press or filter and a high purity separation of thylakoids and liquid phase will be achieved in a later step.

Stabilization Usually there is a stabilization step followed by a stabilization step. This step allows the withdrawal of electron donors such as water molecules that are either bound or not bound to the membrane, to remove activators of the PSII system. Fractions are collected and placed in clean vials. In particular, the first, second and third fractions, which represent the whole extract, the pellet (thylakoid fraction) and the cell debris / membrane fraction, respectively, are lyophilized. The vial is then exposed to vacuum and low temperature (about -20 to -50 ° C) for at least 4 hours. The lyophilized fraction thus remains stable for a long time until water is added to it. Other stabilizing means can also be used. For example, multiple surfactants have been used to confirm their performance to drive out water without compromising the structure of thylakoids. These solvents are: Triton X-100, PEG, beta-D-maltoside, glycine, glycerin, glycerol, TWEEN ™, SDS, LDS, DMSO, cholate, stearate and propylene. Glycol has been used.

Propylene glycol was preferred and would advantageously replace lyophilization as a stabilizer. Propylene glycol not only supplies an inactive, immobile thylakoid preparation (functions and is fully activated by the addition of water, see FIG. 8), but also assists in its solubilization to eliminate thylakoids during hydration. Stabilize. Upon hydration, thylakoids normally form suspensions, but in the presence of 100% propylene glycol, thylakoids form solutions with a solubility limit of 100 mg / mL solution. This solution can be diluted with water for activation. This solvent is also non-toxic.

Optional Additional Fractionation The thylakoid membrane can be further fractionated into fractions. For example, reaction center or photochemical system, light-harvesting complex, cytochrome complex, specific dye (chlorophyll,
Carotenoids), plastoquinones, non-photosynthetic components (cell nuclei), or mitochondria can be predicted.

Thylakoid Integrity and Activity The extracts contain substantially pure (> 90%) thylakoids, they have photosynthetic activity, are stable, and the extracts are controllable. Photosynthetic activity can be achieved by various techniques, including oxygen release (Schlodder et al., 1999), photoreduction of 2,6-dichlorophenolindophenol (DCPIP) (Behera et al., 1983) and fluorescence (Maxwell et al., 2000).
) Has been evaluated. Furthermore, thylakoid integrity has been evaluated with techniques that measure continuous currents, and any disruption should be detected by some change in this current. This current was measured from PSII towards the coupling factor, indicating that the thylakoids contain the major subunits listed above and that they are functional.

When green light is used as the working condition, the dye is stabilized in its basic state (F ₀ ), thus allowing optimization and synchronization of any desired effect. Stabilization is possible because of the abstraction of the primary electron donor. Photosynthetic activity (absent in the quiescent state and appearing upon activation with an electron donor) and stability as measured by chlorophyll and carotenoid concentrations persist for several months after extraction. The chlorophyll / carotenoid ratio is also important for the activity of the complex and for maintaining absorption and consumption of energy.

The extract can be easily detected by its natural fluorescence. Since no toxic products, solvents, detergents and preservatives have been added to the above thylakoids, the product has retained all its original properties. The extract is completely edible. Even if propylene glycol is used to stabilize the thylakoids, the solvent is harmless as it oxidizes to produce pyruvic acid and acetic acid. Currently, this solvent is used as an emulsifier in food products, which means it has surface-active properties (however, it is not detrimental to the integrity of thylakoids). It also has inhibitory activity on fermentation and mold growth. Therefore, the solvent can be used at any step of the method and can be mixed with water during homogenization and not mixed after step c (separation step).

The extract may be provided in the form of a dry to wet solid or in the form of a liquid.
The extract hydrates easily but is sparingly soluble in water, but is completely resuspended in propylene glycol. Thylakoids are reactivated by rehydration. Therefore, it is envisioned that a composition containing solubilized thylakoids will be used and that the thylakoids will be activated upon contact with an aqueous solution.

By-Products Although thylakoids are the products of primary interest in the above procedure, other plant components separated from thylakoids will also be recovered due to their commercial value. The liquid and cell debris / membrane fractions are readily available as starting materials for the isolation of any plant molecule of interest. The latter fraction may be re-extracted to increase the yield of thylakoids recovered per unit plant. It is expected that the components of the other fractions will have superior properties compared to any comparable components obtained from prior art methods. Any other plant component sensitive to ROS may also benefit from this method because the formation of destructive ROS is prevented by the thylakoids prepared according to the present invention or the already formed ROS is trapped. is expected. At all, other components that normally tend to degrade by oxidation will be preserved by removing harmful sources of degradation. Therefore, all plant constituents such as sugars, proteins, lipids, vitamins, minerals and hormones are obtained by fractionating the liquid phase obtained, for example, from the above process, which constituents have a greater specific activity than usual. Will have. In addition to this, appropriate conditioning steps can enrich the components of interest in the extract.

After confirming that the extracts are functional, the next step is to confirm their scavenger activity against ROS.

Uses of thylakoids as antioxidants Antioxidants are compounds that interact with ROS (such as singlet oxygen, hydrogen peroxide, superoxide anions and hydroxyl radicals). Reactive oxygen species may degrade or inactivate other molecules, resulting in harmless degradation products, and cause mutations, cancer or inflammation. They may also be involved in aging. The antioxidant molecules of this extract are: chlorophyll, carotenoids and vitamins (B, C, E, K, ...), Cytochromes and anthocyanins. Thylakoids play a particular antioxidant role because their physical organization allows scavenging of singlet oxygen by carotenoids within their role in protecting chlorophyll. Its quenching effect has both high potency and a relatively long duration, as carotenoids dissipate the energy they enter from triplet chlorophyll as heat and are ready to receive it again. Therefore thylakoids stand out in the field of antioxidants due to their ability to regenerate their functional state, and the tissue structure of the pigment will allow a sustained antioxidant activity.

The thylakoids of the present invention will hereinafter be referred to as FRTS / 1, which constitutes the bioactive molecule complex extracted from plant biomass. The functional antioxidant activity of FRTS / 1 is based on the redox potential of the molecular complex of thylakoids, in which the three-dimensional structure and natural distances between its various pigments and molecules are maintained. ing. This antioxidant is an indicator of optimal structure and optimal composition.

Protein quantification by the fluorescamine method The protein concentration of the stabilized extract is 0.42-0.65 g / g extract.

Antioxidant Function of Thylakoids Main Reaction Pathway in Radical-Induced Lipid Oxidation Naturally antioxidants are used to prevent the oxidation of biomolecules such as proteins, DNA, lipids and the like. Lipid peroxidation is a damaging process, especially in ubiquitous living organisms. Peroxidic radical (ROO ^·) is that they are capable initiate the peroxidation of lipids, and in the becomes an intermediate in a variety of oxidation of biologically important molecules because biological systems Is an important radical.

[Chemical 8] The peroxidation reaction of unsaturated lipid moieties (Scheme 1 to 3) causes a change in its structure,
It ultimately changes the physical properties of the biofilm. Conjugated diene groups are formed during lipid peroxidation (see LOOH in Scheme 3), which is UV / Vi at λ _max = 234 nm.
It has an s absorption (ε = 29,500 M ⁻¹ cm ⁻¹ ), which allows the quantification of the oxidation products formed. To obtain quantitative data, the lipid peroxidation reaction must be initiated in a controlled manner (Scheme 1). To investigate the oxidation of lipids in in vitro experiments, an azo initiator produced a well-defined flux ROO ^· in an aqueous solution that is vented is often used. The most commonly used water-soluble azo initiator is AAPH (Scheme 4, sometimes called ABAP).

[Chemical 9]

The decomposition rate of AAPH at 37 ° C. is k = 1.3 × 10 ⁻⁶ M ⁻¹ s ⁻¹ , and ROO ^·
Efficiency of formation is 50%, that is, AAPH of 1mol produce ROO ^· a 1mol. This method of ROO ^· generation allows a precise amount of calculation of ROO ^·, which are also formed in any time interval.

The most intensely studied antioxidant is vitamin E, and the most active compound of the vitamin Es is α-tocopherol (α-TocH). It is a radical chain scissioning antioxidant that is capable of scavenging two peroxyl radicals to produce a non-radical product, thereby blocking lipid peroxidation (Equations 5 and 6). ).

[Chemical 10]

[0114] tocopheroxyl radicals formed in Scheme 5 ^(Toc-) are relatively non-reactive radicals, usually can not propagate the radical oxidation chain reaction. As soon as all TocH is consumed, lipid peroxidation occurs as if no antioxidant were present.

Carotenoids (Cars) are the most likely “antioxidants” of FRTS / 1, because chloroplasts utilize carotenoids such as carotene and xanthophylls for their exciton-propagation chain. There are several pathways through which carotenoids can react with ROS, and their overall behavior ranges from their antioxidant activity to their ineffectiveness with respect to loose lipid peroxidation. The experimental results obtained depend on the reaction conditions used. Possible reaction of the carotenoids and ROO ^· is,

[Chemical 11] Is.

Some reactions lead to the formation of non-radical products (reactions 7 + 8, 10
+11, 12 + 14), resulting in an overall antioxidant behavior of carotenoids. Other reactions have generated peroxyl radicals (Equations 9 and 13) and may be involved in the propagation of lipid peroxidation reactions. The overall behavior of carotenoids cannot be clearly predicted due to the various possible reaction paths.

Depending on the possible reaction path of the antioxidant used, the temporal profile of the concentration of LOOH detected shows certain characteristics. The amount of ROO. Generated by using an azo initiator as a peroxyl radical source can be calculated ^and the amount of lipid oxidized can be determined from the 234 nm absorption of the conjugated diene moiety. The amount of ROO ^· trapped between the lag phase can also be determined.

The method in FRTS / 1 may "restore" the "native" antioxidant properties. A possible mechanism for such behavior may be electron transfer rather than radical scavenging process (see equations 15 and 16), for example:

[Chemical 12] Is.

The concentration / time profile of lipid oxidation in the presence of FRTS / 1 makes it possible to test this hypothesis. Possible lag phase duration between lipid peroxidation experiments (Fig. 9).
), The amount of “scavenged” radicals can be calculated. This makes it possible to draw the conclusion that FRTS / 1 can inactivate the peroxyl radical. However, it should be noted that the possible regeneration of the antioxidant properties described can only be effective as long as FRTS / 1 is able to exert its full "natural" activity. Overall, the experiments will eventually provide quantitative data on the antioxidant capacity of ERTS / 1 (see Figure 10 for the paradigm of antioxidant kinetics of FRTS / 1 compared to Trolox). When β-carotene is compared to Trolox, the former is an antioxidant but lacks the lag phase normally associated with antioxidants.

Since lipids are a major component of cell membranes, lipoproteins and other membrane structures of living organisms, PLPC-FRTS / 1 (1-palmitoyl-2-linoleoyl-s was used in this study.
N-glycero-3-phosphatidylcholine) micelles were used as substrates for standard oxidative analysis. PLPC introduced by peroxyl radicals generated from the initiator 2,2'-azobis- (2-amidinopropane) dihydrochloride (AAPH)
-FRTS / 1 oxidation results in the formation of a conjugated diene system with an absorption maximum at 234 nm with the oxidation of the linolenic acid moiety to the corresponding hydroperoxide.

Preparation of PLPC (1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine) micelles 25 mg / mL PLPC-FRTS / 1 solution in CHCl ₃ (Avanti Polar Lipids
) Was evaporated to dryness in a stream of N ₂ . C in advance to remove metal ions
PL buffered phosphate buffered saline (PBS) (281 μL) treated with helex (registered trademark)
In addition to PC-FRTS / 1, this mixture was vortexed for 2 minutes. 109 μL of sodium cholate aqueous solution treated with Chelex (registered trademark), 30 mg / mL (Aldrich Chemic
al Company Inc., Milwaukee, WI, USA) was added to this mixture and vortexed for 2 minutes. This mixture was passed through a polycarbonate membrane (pore size 100 nm) 20 times to homogenize the size of the micelles.

FRTS / 1 was dissolved in CHCl ₃ (12 mg / mL) and 1 mL of this solution was added to 6 mL of PLPC-
By mixing with FRTS / 1 (25 mg / mL), PC-FRTS / 1 with a final concentration of 6 mg / mL was obtained.
The micelles were prepared as described above. The azo initiator (AAPH) was dissolved in PBS to a concentration of 5 mg / mL before use.

Normalization Initial experiments were performed using the dehydrated form of FRTS / 1 to standardize micelles, azo initiators and wavelengths. Optimal absorption was obtained at 234 nm.

5 mg / m of PLPC-FRTS / 1 and PLPC-FRTS / 1 + FRTS / 1 dissolved in 3 mL of PBS
100 μL prepared by diluting in duplicate with L azo initiator (10 μL and 20 μL)
Two preliminary experiments were carried out on the Cary 3 UV / Visible spectrophotometer from Varian at 37 ° C. for 10 hours at a wavelength of 234 nm. 234nm due to PC-FRTS / 1
The background absorption at was too high under these reaction conditions.

Experiments Based on the results obtained in the above experiments, it was decided to use a further diluted FRTS / 1 solution (final concentration of FRTS / 1 was 6.7 μg). AA as a negative control
Compound 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) resistant to oxidation mediated by peroxyl radicals derived from PH
FRTS / 1 was incorporated into the micelle prepared in (1). 15.937 mg DMPC-FRTS / 1 (stock solution 25 mg / mL) +1.053 m dissolved in 0.6375 mL PBS
L PBS + 0.408 mL sodium cholate + 0.2435 dissolved in CHCl ₃
Micelles were prepared as described above from mL FRTS / 1 (2 mg / mL). Therefore, the following experiment was carried out. 3 mL PBS + 100 μL PLPC-FRTS / 1 micelle + 10 μL azo initiator 3 mL PBS + 100 μL PLPC-FRTS / 1 micelle + 20 μL azo initiator 3 mL PBS + 100 μL PMPC + FRTS / 1 + 10 μL azo initiator 3 mL PBS + 100 μL PMPC + FRTS / 120 μL azo initiator · 3 mL PBS + 10 μL azo initiator · 3 mL PBS + 20 μL azo initiator

[0126]   The reaction was spectroscopically monitored at 37 ° C. for 10 hours.

Conclusions These results show that the maximum O of PLPC-FRTS / 1 micelles containing FRTS / 1 at 0.3 mg / mL.
DPC (234 nm) became 0.25 after 180 minutes, whereas PLPC without FRTS / 1-
The OD of the FRTS / 1 micelles is 3.2, which indicates a high temperature after the same amount of time. These results arguably show that FRTS / 1 exhibits antioxidant properties.

Antioxidant Properties of FRTS / 1 Solution Compared to Vitamin E To compare the antioxidant properties of FRTS / 1 and Trolox, water soluble vitamin E analogs, a low concentration of antioxidant was used (in PBS. Dissolved in 0.3 mg / mL). The following samples were run on a spectroscope for 10 hours at 37 ° C. 1.3000 μL PBS + 10 μL azo initiator 2.3000 μL PBS + 100 μL PLPC micelle + 10 μL azo initiator 3.2998 μL PBS + 2 μL antioxidant (0.5 mg / mL aqueous solution) +10
0 μL PLPC micelle + 10 μL azo initiator 4.2995 μL PBS + 5 μL antioxidant (0.5 mg / mL aqueous solution) + 10
0 μL PLPC micelle + 10 μL azo initiator 5.2990 μL PBS + 10 μL antioxidant (0.5 mg / mL aqueous solution) +1
00 μL PLPC micelle + 10 μL azo initiator 6.2980 μL PBS + 20 μL antioxidant (0.5 mg / mL aqueous solution) +1
00 μL PLPC micelle + 10 μL azo initiator 7.2990 μL PBS + 10 μL Trolox + 100 μL PLPC micelle + 10 μ
L azo initiator 8.2980 μL PBS + 20 μL Trolox + 100 μL PLPC micelles + 10 μL
L azo initiator 9.2970 μL PBS + 30 μL Trolox + 100 μL PLPC micelle + 10 μL
L azo initiator

[0129]   FIG. 10 shows that FRTS / 1 is more persistent than Trolox.

Protective Mechanisms Radical oxygen species (ROS) readily interact with intracellular macromolecules and structures, resulting in altered membrane permeability, protease and nuclease activation, and altered gene expression (Yu , 1994; Schiaffonati and Tiberio, 1997). ROS
It is well known that these intracellular alterations introduced by PAT lead to cell death by apoptosis. The authors planned the following: -To evaluate the antioxidant properties of FRTS / 1. -To determine the mode of action of FRTS / 1 as an antioxidant.

IMR-32 cells constitute an excellent model for assessing the antioxidant capacity of our extracts. These cells are neuroblastoma cells that are sensitive to the oxidative stress that causes apoptosis.

Experiment 1. Standardization of experimental conditions Selection of cell culture A human neuroblastoma cell line (IMR-32) (Kim et al., 1999) known to respond to oxidative stress by apoptosis was used as an in vitro cell model. IMR-32 cells are particularly sensitive to ROS and other toxins because their p53 gene product is trapped in the cytoplasm. This confinement inactivates p53 even if the gene is not mutated.

Selection of ROS Inducers Tertiary butyl hydroperoxide (TBHP) was used as an oxidative stress inducer. TBHP has no neuronal specificity in contrast to oxidative stress induced by MPTP in dopaminergic neurons. This allows comparisons when studies related to more generalized oxidative stress conditions (such as those found in many neurodegenerative diseases) need to be performed with different cell phenotypes. Ah

A. Determination of optimal dose of TBHP to induce apoptosis in IMR-32 cultured cells Experiments were carried out using 1000 IMR-32 cells per well with 1 hour incubation. TBHP at 50, 75 and 100 μM is 78%, 87% and 8
It resulted in 7% apoptosis. 50 μM to induce apoptosis in other experiments
I chose TBHP.

B. FRTS / 1 Administration Various dilutions were used with TBHP on IMR-32 cells. A lyophilized thylakoid fraction was used as a starting material to form a 1:10 matrix solution in propylene glycol. Unless otherwise specified, the dilutions stated are the dilutions of this matrix solution. The protection provided by FRTS / 1 is 1:10, 1: 100 and 1: 1 dilution
000 was 28%, 35% and 75%, respectively. The latter dilution was applied for further experiments.

1. Cell Culture IMR-32 cells were grown in MEM supplemented with 10% FBS at 37 ° C. in a humidified atmosphere of 95% air and 5% CO ₂ . Cells were seeded at a density of 1 × 10 ⁴ cells / T25 Falcon tissue culture flask and subcultured twice a week. Cultures after 48 hours were used for all experiments. In the first experiment, 100 per well
0 cells were seeded. The number increased to 3000 as shown later in the table.

2. Oxidative stress in the in vitro protocol All wells except all 12 rows of 96 well plates (Linbro flat bottom) were seeded with 1000 or 3000 cells / well / 100 μL. After 24 hours, the cells were washed twice with 250 μL of PBS (pH 7.2) and 32 wells each containing 100 and 200 μM TBHP solution (obtained from Sigma Chemical Company 7
0% aqueous solution) for 1 hour each. After 1 hour, cells were gently washed twice with PBS prior to adding fresh growth medium. After 24 hours, viable cells were analyzed by a high-sensitivity fluorescence analysis method based on the DNA-binding fluorescent dye Hoechst by the following method. This test measures the total DNA of the population, and the measurement value correlates closely with the cell number. The medium was aspirated by gentle aspiration. 250 μL of cells
Rinse with PBS. PBS was aspirated with gentle aspiration. Repeated the rinsing steps. 100 μL in each well except row 12 DNA standards and blanks
Lysis buffer (0.02% SDS in 1 × SSC) was added. The plate was occasionally incubated for 1 hour at 37 ° C with vortexing. 40 μg / mL
Was added to the DNA wells and 100 μL of 1 × SSC buffer was added to the wells treated as blanks. 100 μL of 40 μg / m dissolved in 1 × SSC
L Hoechst 33258 was added to each well and covered with aluminum foil for protection from light. The plate is gently agitated for 5 minutes and the excitation wavelength 355
Fluorescence was read at nm and emission wavelength of 460 nm.

3. Calculation Untreated CT = 100%, survival after TBHP: 42%, dead cells: 100-42 =
58%, aggregate size after PC: 88% Difference from CT: 100-88 = 12% TBHP
Expected set size after + PC: 100-58-12 = 30%. Recovery survival: 60% survival gain%: 60-30 = 30 exertion protection: 30: 60 × 100 = 50%.

4. Evaluation of cytotoxicity by LDH analysis For this experiment, IMR-32 cells were grown in MEM medium without l-glutamine, phenolphthalein and sodium pyruvate and 3000 IMR-32 cells / well in 96 wells. Planted in a plate. Twenty-four hours after oxidative stress, cells were washed twice with PBS and two rows were treated with PC-FRTS / 1 at 1: 1000 and 1: 10000 for each treatment at 37 ° C. After 1 hour, cells were washed twice with PBS and PBS was replaced with growth medium. Two rows were treated with PC-FRTS / 1 at 1: 1000 and 1: 10000 respectively, while two rows were treated with 25 and 50 μM TBHP respectively. Two rows remained untreated as a control. LDH activity is a Lactate Dehydrogenase Assay supplied by Sigma Diagnostics
Measured by Kit. Colorimetric analysis measures residual pyruvate, a substrate for enzymatic reactions. The basal activity present in the solution alone (without cells) is considered to be 0%,
It was systematically subtracted from each experimental value. Two rows of untreated controls were Triton X
Decomposed at -100 (0.02%) and treated as a sample with 100% LDH release.

The first two experiments were performed to standardize the procedure, number of cells used, optimal absorption wavelength, optimal pyruvate substrate used. It was established that 3000 cells / well, 0.4 mL pyruvate, spectroscopic reading at 440 nm was the standard.

[0141]   FRTS / 1 was used in oxidative damage with pre-treatment, co-treatment and post-treatment.

[0142] Preprocessing   Cells were pretreated with FRTS / 1 for 2 hours before exposure to TBHP administration.

[Table 2]

[0143] ・ Simultaneous processing   Cells were treated with FRTS / 1 and TBHP simultaneously for 1 hour.

[Table 3]

[0144] ・ Post-processing   Cells were treated with 3 doses of FRTS / 1 one hour after exposure to TBHP.

[Table 4]

“Cell damage rate on TBHP / CT” describes the damage caused by oxidants and is calculated as a percentage from the viable number of untreated controls. "Rate of reduction with FRTS / 1" refers to the difference in aggregate size between control and FRTS / 1 exposed cells. “Expected total reduction” is the sum of aggregate size differences due to individual exposures of TBHP and FRTS / 1. "Expected survival rate" is 100-the expected reduction rate in total. "TBHP + PC / CT viability" is the actual viability in the presence of FRTS / 1 diluted 1: 1000. "PC
The "protection ratio exerted by" is calculated as described above.

In the pretreatment experiments, the effect of FRTS / 1 is increased compared to the protection exerted by the posttreatment (50% for 23% and 77% for 62%). PC-FRTS /
It is strongly suggested that the protective effect of 1 is exerted through its antioxidant properties. At low dose concentrations (25 μM), there is double protection against damage caused by oxidants.

Clearly, the efficacy of PC-FRTS / 1 is confirmed as a preventative treatment due to its antioxidant properties.

The antioxidant properties of FRTS / 1 are confirmed in co-treatment experiments. The efficiency of this product is comparable to that reported with the post-treatment at the highest doses of oxidant and intermediate to that obtained with the pre- and post-treatments respectively.

FRTS / 1 exerts protection against apoptosis induced by TBHP during TBHP injury.

The strong antioxidant properties of FRTS / 1 are confirmed through its protective effect against ROS generated in IMR-32 cells by the oxidant TBHP. Under our standard conditions, the protective effect averages 62%.

[0151]   Antioxidant effects occur with pre, simultaneous and post treatments. (Figs. 11a and b)

FRTS / 1 exerts even greater protection as the oxidative damage caused by TBHP to IMR-32 cells increases (TBHP dose increases). This is independent of the concentration of FRTS / 1, because 1/1000 as well as 1/1000
As seen at a dilution of 10,000 (shown in Figures 12A and 12B, respectively). These results show the vitality of this extract.

The fact that protection increases with increasing damage indicates that the mechanism of action of FRTS / 1 may differ from that of conventional antioxidants as shown by studies on reaction chemistry, 1) The protective effect exerted by FRTS / 1 lasts longer, 2) Vitamin E and its analogues are used at the given concentrations and exert their antioxidant effect. It does not appear to be the case in ERTS / 1 exemplified by the above chemistry.

The correlation between the amount of oxidative damage and protection by PC-FRTS / 1 is a unique property exhibited by this antioxidant.

As the cell density increases from 1000 to 3000, the dose of product received by each cell is clearly reduced. This is a well-known effect in toxicology and pharmacology. The protection exerted by FRTS / 1 remains excellent even when the number of cells to be protected is tripled. The effect is reproducible.

Evaluation of Apoptosis of IMR-32 Cells by Lactate Dehydrogenase (LDH) Assay The preexisting LDH assay measures the release of LDH enzyme by apoptotic cells. The assay used in this study uses a colorimetric reaction to measure the residual amount of the enzyme substrate (pyruvate). The more substrate in solution the less released enzyme. The medium cells were set to 0% enzyme activity release and the decomposed cells in the medium were set to 100% release. The numerical values in the table are calculated from these two parameters. Extent of apoptosis measured by LDH release by damaged cells

[Table 5]

The total LDH release by individual exposure of TBHP + FRTS / 1 was compared to the observed LDH activity (last row).

The protective effect of FRTS / 1 is clear. Post processing by FRTS / 1 occurs by TBHP
Effectively protects IMR-32 cells against ROS-induced apoptosis.

[0159] Conclusion   FRTS / 1 compounds show strong antioxidant properties as assessed by chemical analysis.

Biological in vitro analyzes also show these highly efficient antioxidant effects: • The chemically shown antioxidant properties of FRTS / 1 are biologically confirmed • FRTS / 1 shows a highly protective effect against ROS damage that causes apoptosis of IMR-32 cells after TBHP exposure, and FRTS / 1 exerts a higher protection effect as oxidative damage increases. (RO
It presents unique dynamic properties such that the higher the damage by S, the higher the protection by FRTS / 1), and FRTS / 1 exhibits a long lasting antioxidant effect (for many hours), which is stable Unique to this antioxidant, it shows a high degree of degree and / or regenerative capacity, and FRTS / 1 is efficient at non-toxic doses.

Cell-Cell Interactions In order to be an effective medication, the extract must satisfy at least some properties. Among other things, the extract is not toxic, immunogenic, or interferes with normal tissue function, especially the transport of oxygen and carbon dioxide by red blood cells.

The extract must be dispersed in the subject's body, targeting the tissue or organ to be treated, but not sticking to red blood cells. The authors confirmed whether macrophages, the first line of defense cells, did not destroy this extract. The mode of destruction of macrophages is usually the production of free radicals that destroy large particles before phagocytosis. Upon phagocytosis, macrophages produce cytokines, which are molecules that signal the presence of some invaders or tissue dysfunction. Cytokines are molecules that are sent to other cells and signal the presence of invaders or tissue dysfunction.
Cells that respond to cytokines are cells such as fibroblasts, endothelial cells, macrophages, lymphocytes, neutrophils and eosinophils. These cells are involved in the process of tissue and organ destruction and / or reorganization. These processes include inflammation. If this response is disrupted or if invaders are continuously present in the organism, the latter suffer chronic diseases such as rheumatism, dermatitis, conjunctivitis, alveolitis, asthma, and cancer.
The interaction between cells and FRTS / 1 is performed with FACS technology. This device also detects the fluorescence of the cells (autofluorescence) and any fluorescent molecules immobilized around them. FRTS / 1
Is an autofluorescent complex and therefore the interaction can be quantified without any modification.

Conclusion: FRTS / 1 was mast cells (positive control, RCMC supplied by ATCC, at 37 ° C).
) Compared to macrophages (commercially available strain: NR-8383 supplied by ATCC)
Stick to. Macrophages phagocytose FRTS / 1 (1/3) after 2 hours, which is FR
It shows that TS / 1 stays in the bloodstream for a long time. Slow phagocytosis is not bad news, but vice versa, because another type of beneficial effect can be observed following activation of cytokines (the "Phase II" effect). Since macrophages phagocytose this extract and IMR-32 cells appear to allow this extract to enter the cytoplasm, it is thought that this extract can enter cells by endocytosis.

Ex Vivo Models of Oxidation The liver and brain perfusion models are excellent experimental models in response to oxidative stress. They were used to show the protective effect of this extract on living organs.

Liver Perfusion Model Multiple liver functions were evaluated. The method used to perfuse the liver was Drouin et al., 2000.
, And Lavoie et al., 2000. Glucose, lactate, ALT, and LDH were determined spectroscopically, while bile formation was measured simply by volume. There was no adverse effect observed with perfusion of this extract when compared to the control vehicle. In contrast, the efficacy of the extract in reducing the expression of stress caused by ischemia, followed by reperfusion (I / R), protects it against oxidative damage induced by the liver. It was the result of having.

In this model, the liver is perfused in situ with a controlled extracorporeal circulation that keeps the vascular bed intact and preserves its structural and functional integrity but is separated from the liver. This model is well documented (Ross, 1972) and allows the study of cellular damage as well as oxidative stress (Bailey, 2000).

The effect on liver survival is designed to assess the effect of FRTS / 1 on liver viability. More specifically, the effect of FRTS / 1 on liver function during in situ perfusion will be assessed.

Sprague-Dawley rat livers are perfused in situ in a standard Krebs-Henseleit (KH) solution in a single pass system. [PH7.4, O ₂ : CO ₂ (95%: 5%)] KH solution is
118mM), KCl (4.8mM), KH 2 PO 4 (1.2mM), MgSO 4 · 7H 2 O, CaCl 2 (1.5mM), NaHCO 3 (25m
M) and albumin (2% w / v).

Under anesthesia (pentobarbital; 50 mg kg ^-1 body weight), a laparotomy is performed to expose the portal vein, inferior vena cava and biliary tract for cannulation. Cannulation into the portal vein is used to pump perfusate into the liver, and into the vena cava is used to collect perfusate at the liver exit. Cutting the vagus nerve to isolate the liver from the effects of any vasomotor nerves. The total surgical procedure is completed within 15 minutes. The time interval between cannulation of the portal vein and initiation of circulation does not exceed 3 minutes. The time course between cardiac arrest caused by a thoracotomy and the start of perfusion does not exceed 1 minute.

The total duration of perfusion was 60 minutes. Rinsing was done during the first 30 minutes. During that period, glucose (8 mM) and lactate (0.5 mM) were added to the KH solution (37 ° C).
mM), alanine (0.2 mM) and glycerol (0.02 mM) were added and circulated to the liver in an open circuit system. The livers were then exposed to vehicle alone (control) or FRTS / vehicle (FRTS / 1 treated group) for another 30 minutes. This vehicle contains either saline (1 mL / 800 mL perfusate) or 1,3-propanediol (24 mL / 800 mL perfusate). Add FRTS / 1 to propanediol (0.06 mg FRTS / mL propanediol: 24 mL / 8
00 mL perfusate) or in addition to saline (2 mg FRTS / mL saline: 1 mL / 800 mL perfusate). Flow rate of perfusate is 6 mL / min / 10
It was kept constant at 0 g body weight. Small samples of perfusate were taken at the inlet and outlet of the liver to determine the production of metabolite utilization.

The concentrations of glucose, lactate, ALT and LDH in the perfusate were determined by Sigma-Aldrich Cana
D an. 17-UV, No. 735, No. 59-UV and No. DH1240-UV, determined by spectroscopy using the commercially available techniques disclosed in each. Substrate production or extraction by the liver is measured by integrating the perfusate flow rate with the difference between the metabolite inlet and outlet.

The results were combined as the vehicle properties did not affect the measured parameters.

Bile production was similar in both control and treated groups (0.55 ± 0.10 v. 0.62 ± 0.12 mg / min / g liver in control and treated groups, respectively). During reperfusion, bile production was attenuated in both treated and control groups compared to preischemic levels. Bile production returned to normal levels within 10 minutes after reperfusion.

At the inlet, glucose concentrations were similar in both groups (7.04 ± 0.40 v. 7.24 ± 0.07 mM in control and treated groups, respectively). In both groups, the liver tends to utilize glucose slightly. Exposure to FRTS / 1 has no effect on glucose uptake (0.3% each for control and treated groups).
2 ± 0.21 v. 0.39 ± 0.25 μM / min / g liver). The lactate concentration at the inlet was also similar in both groups (0.60 each for control and treated groups)
± 0.33 v. 0.50 ± 0.10 mM). Exposure to FRTS tended to produce slightly lactate in the treated liver (in the control and treated groups respectively −
0.01 ± 0.1 v. 0.40 ± 0.005 μM / min / g liver).

Liver perfusion itself does not cause any release of ALT or LDH in the control group (−0.07 ± 0.14 and 1.79 ± 0.47 μM / min / g respectively). FRTS
Treatment with 1/1 did not appear to cause the release of ALT or LDH although it tended to increase somewhat (0.57 ± 0.21 and 2.36 ± 1.10 μM / min / g
). Partial perfusion of the liver prior to using FRTS had a progressive increase in LDH production by the liver (35.57 ± 8.96 μM / min / g).

These preliminary results suggest that FRTS / 1 has no significant effect on the viability of the perfused liver. More specifically, FRTS / 1 treatment does not appear to alter liver function during 30 minutes of perfusion, as assessed by glucose utilization, lactate and bile production. There is no apparent structural damage to hepatocytes as there is no increase in ALT and LDH.

[0177] The above was slightly modified to study the effects of ischemia and reperfusion. The total perfusion was 105 minutes. The washout period was configured until the 31st minute. During that period, 8 mM glucose and 0 lactate were added to a KH solution (pH 7.4 and O ₂ : CO ₂ 95%: 5% present).
． 5 mM, alanine 0.2 mM and glycerol 0.2 mM were added. The solution was circulated in an open circuit system. The liver was then exposed to this extract or vehicle: 1,3-propanediol for 15 minutes. Perfusion rate is 6 mL / min of 100 g body weight
Was kept constant (Drouin et al., 2000, Lavoie et al., 2000). Perfusion was stopped for 30 minutes. During this arrest period, ischemia developed. Then, perfusion was restarted for a duration of 30 minutes. Perfusate was collected at the inlet and outlet of the test liver to determine the production or utilization of the biological marker to be evaluated.

Upon reperfusion, biosynthesis is attenuated compared to pre-ischemia in both control and treated livers. These levels return to normal within 10 minutes after initiating reperfusion. Control and treated livers release glucose in the same manner (10.4 ± 0.7 μM · min− ¹ ·
g ^-1 ). However, glucose production was slightly higher after 80 minutes in the treated group compared to control organs. Lactate was added during ischemia to control organs (4.5 ±
0.1 μM · min ⁻¹ · g ⁻¹ ) compared to 0.1 μM · min ⁻¹ · g ⁻¹ ) in the treated liver.
9 ± 0.5 μM · min ⁻¹ · g ⁻¹ ).

Pretreatment with this extract reduces the release of ALT during reperfusion (1.
09 ± 0.44 mU · min ⁻¹ · g ⁻¹ vs. 2.44 ± 0.79 mU ・ min ^-1・ g ^-1 )
. Pretreatment with this extract appears to attenuate the ischemic shock during the first 30 minutes of perfusion. Ischemia without any pretreatment with this extract causes an increase in LDH during reperfusion (108.7 ± 27.3 mU · min ⁻¹ · g ⁻¹ ). LDH pretreatment with this extract
(115.9 ± 60.8 mU · min ⁻¹ · g ⁻¹ ).

Plasma concentrations of potassium and sodium were also measured in both groups. At the beginning of reperfusion, potassium release in the treated group is above control (0.43 ± 0.01 mU · min ⁻¹ · g ⁻¹ vs. 5.4 ± 0.
¹ mU · min ⁻¹ · g ⁻¹ ). In the entrance perfusate, plasma levels of potassium and sodium were similar in both groups (K ⁺ = in control and treated groups, respectively).
5.6 ± 0.3 mM and 5.4 ± 0.1 mM; control and treated groups respectively
Na ⁺ = 142.2 ± 8.6 mM and 137.2 ± 15.2 mM).

At the beginning of reperfusion, sodium release was also higher in the treated groups when compared to controls (1.2 ± 1.1 μM · min ⁻¹ · g ⁻¹ and 16.5 ± 3.
9 μM · min ⁻¹ · g ⁻¹ ).

Ischemia prior to reperfusion is characterized by impaired circulation and metabolism, and tissue damage caused by free radicals (Lee, 2000). This particular model is
Currently used to assess free radical-induced damage in transplanted tissues or organs (Smrekova 2000, Cohen 2000 and Hachimoto 2000.
). Structural and functional disorders are caused by I / R and increase enzyme release (Bai
ley 2000, Vollmar 1994 and Perlata 1999), Reduced bile production (Vollmer 1994)
) And depletion of ATP storage (Hwang 1999 and Amllet 1990). Ischemia can lead to the release of singlet oxygen, peroxides, and superoxide (O _2- ^· , H ₂ O ₂ , OH ^· ) following the release of metal ions such as iron and copper. Yes (Halliwell 1999). Therefore, this model is excellent at characterizing the protective effect in the presence of the extract, if any, following radical damage.

The metabolic changes that ensure hepatocyte protection are affected by pretreatment with this extract. The resulting radical attack of I / R causes a decrease in the intracellular content of ATP, which changes the energy state of the cell (Peralta 2000, Ma
llet 1990). Tamarina et al. (1984) suggest that ischemia damages the cell's glycolytic system, which makes lactate production difficult. Healthy cells (or cells under the action of protective agents) protect themselves from such stress by increasing glycolysis-mediated ATP production. Sano et al. (1995) proposed that activation of glycolysis reduced I / R-induced free radical formation. Phosphoenolpyruvate also prevents ATP loss resulting from I / R (Saiki 1999). Hwang etc. (1
999) suggest that reducing the NAD ⁺ / NADH ratio is essential for resistance to free radicals in order to minimize cell damage. Pretreatment of cells with this extract increases lactate production during ischemia, which clearly reflects activation of protective mechanisms against ischemia-induced ATP reduction. Kowalski 1992 and Groussard 200
0 suggests that lactate may play a protective role in ischemia. Lactate can be buffered superoxide (OH ^·), which is also peroxide occurs pyruvate and buffers the superoxide, while decomposing into acetate and CO ₂ (Herz 1997).

The potassium efflux of cells pre-treated with this extract also supports the protective effect of this extract on cells. Peroxidation of membrane components can damage potassium channels (Halliwell 1999). Potassium release may be a beneficial match to metabolic stress (Wang et al., 1996). Opening of potassium channel is intracellular ATP
It will allow substrate capture for production (Wondergem 1980). Potassium release also HCO ₃ ^- transport also inhibited, and contribute to the acidification of the cytoplasm, it will also become protection to cells (Currin 1991). Potassium release appears early in the perfusate, causing ALT
It precedes release and is proportional to ATP decrease (Mets 1993).

Intracellular sodium accumulation plays a major role in I / R-induced cell damage (Carini 2000, vanEchteld 1991 and Xia 1996). This increase is 1) AT
May be due to Na ⁺ / K ⁺ pump dysfunction due to decreased P and increased inorganic phosphate, or 2) stimulation of Na / H anticarriers due to cytosolic acidification (Zhao-fan 199
6). Any reversal of such a situation could reduce the impact of sodium accumulation-induced metabolic stress (Fiegen 1997). Therefore sodium release may be a protective mechanism against damage. A decrease in both potassium and sodium was observed in liver perfused with this extract, supporting the protective effect of this extract on hepatocytes. Ten minutes after reperfusion, sodium release was greater in the treated group compared to the control group, suggesting a slow recovery of TP content in the control.

The extract stimulates intracellular mechanisms associated with cell protection against radical attack.

Ex vivo effects of this extract in the brain Many brain lesions are associated with increased free radical production. The latter is believed to contribute to the process of neuronal tissue degeneration, ie after cerebrovascular disease or during the development of Alzheimer's disease. It is considered that the anti-radical component may be useful for reducing the expression of neuronal tissue degenerative diseases, or preventing or treating them.

The hippocampal region of the brain is vulnerable to the neurotoxic effects of free radicals, cerebral anoxia. It has been shown in vitro that neurotransmission is attenuated during anoxia due to overexpression of antioxidant molecules. Therefore, the effect of this extract on hippocampal neurotransmission was investigated. An electrophysiological response was registered in this brain tissue. In particular, the recovery of nerve cell potential was investigated. A 450 μm-thick hippocampal slice section was taken from the rat brain and transferred to the electrophysiology space. These sliced sections were kept for 60 to 90 minutes in an oxygen-enriched physiological solution with or without the concentration of the extract.
After this rest period, electrical stimulation was applied to the central hippocampus (Schaffer, CA1 region) every 25 seconds. These electrical stimuli evolved synaptic responses. To quantify the synaptic transduction potency after anoxia, the initial slope of the post-excitation potential was calculated. To examine the nerve cell potential, a high frequency stimulation train was applied to the neural circuit after anoxia. As a positive control, it is possible to measure the response to glutamate in ischemia or non-ischemia and the presence of this extract should restore the response to glutamate.

FRTS / 1 Toxicity Toxicity was evaluated in two models: in situ liver perfusion and in situ brain perfusion. These organs showed no signs of toxicity due to the presence of this extract.

Side Effects This Extract is Non-Immunogenic The immune response elicited by this extract was investigated by intraperitoneally injecting mice 125 μg of this extract three times at 7 day intervals. One week after the last infusion, blood was collected for immune serum attention. Blood samples were immediately placed on ice. Clotting was allowed to proceed on ice and samples were centrifuged 12 hours after collection. The centrifugation conditions were as follows: 2500 g for 10 minutes, which yielded about 500 μL of serum. The extract was adsorbed onto microplates to provide the immobilized antigen preparation. 200 μL of this extract (5 μL
g / mL) and 200 μL of ELISA buffer were added dropwise to the wells of a 96-well plate. The ELISA buffer was made with 100 mM sodium carbonate buffer (pH 9.6). After incubating this antigen overnight at 4 ° C., the non-adsorbed antigen was removed by washing with sodium phosphate buffer 100 mM / NaCl, 100 mM (pH 7.4) three times. Free adsorption sites were blocked by incubation with a 3% casein solution in sodium phosphate / NaCl buffer for 60 minutes at room temperature. Excess casein was washed off 3 times. Antibody-supplied serum samples, if any, were prepared as follows. Ie, serially diluted with sodium phosphate / NaCl buffer supplemented with 0.05% Tween 20 ™, 25 μL of these dilutions
Was added to the wells. The microplate was further incubated for 1 hour at 37 ° C, followed by 5 steps with sodium phosphate / NaCl / Tween buffer following this step.
Washed twice.

The presence of antibody was revealed by the formation of anti-Ig-peroxidase complex. Enzyme diluent 2 diluted with sodium phosphate / NaCl / Tween buffer in each well
5 μL was added followed by incubation at 37 ° C. for 1 hour. Enzyme dilutions were made using conjugates (these were peroxidase-labeled anti-Ig goat antiserum, IgM, IgG, Ig1
, Ig2 and Ig3), and varied between 1: 750 and 1-3000. After the labeling step, the plate was washed 5 times. 100 mM phosphate citrate buffer (
0.015% hydrogen peroxide as a substrate dissolved in pH 4.0) and 0.05% chromogen ABTS (2,2 azinodiethylbenzthiazoline-6-sulfonic acid) were added to the wells. Incubation proceeded at room temperature in the dark for an additional 30 minutes. By the action of the enzyme,
A spectroscopically readable colored substrate was released with an absorption wavelength of 405 nm. The serum level of antibodies specific for this extract should be proportional to the color intensity. The results obtained showed that this extract was not immunogenic for the test individual.

These results indicate that this extract is non-toxic to individuals as it is neither liver toxic nor immunogenic.

Composition and Medication Therapy Due to the stability and potency of the extract and the fact that it is non-toxic to animals,
It is believed that doses of 1 ng / kg body weight to 1 g / kg body weight per day can be administered to an individual in need of such administration (preferably in the lower μg range). The dose will depend on the aggressiveness required to treat the disease. The dosage will also depend on the formulation and the route of administration. For example, a topical composition will not have the same dosage as an intravenous composition or an enteral composition.

Topical Compositions for the Treatment of Skin or Mucosal Diseases Allergy / Asthma Brown Norway rats have high IgE production. There is a well-established model ¹ of allergen-induced airway hyperreactivity in Brown Norway rats, which is an early and late (70% of animals) phase response, increased antigen-specific IgE after active immunization. ² reflects many features of human allergic asthma, including airway inflammation ³ , and increased bronchial responsiveness to several stimulants after allergen challenge.

[0195]   Measurement of pulmonary arterial response: Brown Norway rat was previously reported^FiveAs menstruation
1mg ovalbumin / 100mg Al (OH) dissolved in saline₃1 mL intraperitoneal injection
Sensitize by shooting. 21 days later, animals were anesthetized and reported previously⁶So
Intubate the end of the endotracheal tube attached to the Plexiglas box. Pressure transformer
Changes in pleural pressure using a water-filled esophageal catheter attached to a deducer
To judge. The airflow is coupled to a differential transducer mounted on the Plexiglas box.
It is measured by a combined respirometer. Airflow, volume and pressure difference between lungs, lung resistance (R _L ) Is determined at different times to distinguish both early and late phase responses.
The sensitized Brown Norway rat is Roxon Medi-Tech Lte (Montreal, PQ).
Using a "Wright" nebulizer obtained from
Saline also using compressed air that enters the Plexiglas box at a forceful pressure
Attack with ovalbumin (dissolved in saline to 2%). Lung resistance is the first one
The interval is measured every 5 minutes, and the following 10 hours are measured every 15 minutes. Intraperitoneal injection or suction
Pre-, simultaneous and post-treatments with this extract administered in groups (about 1 to 100 μg)
It will be tested with this model to provide improvements.

Protection Against UV Irradiation FRs Prevent or Reduce UV-induced Skin Damage in Hairless Mice
I investigated the ability of ST / 1. Since most skin cancers are known to be triggered by exposure to UV radiation, there is a need to identify new potential natural compounds that can prevent the harmful effects of UV radiation.

Animals Hairless, albino mice (SKH / 1) were purchased from Charles River laboratories (Wilmin
gton, MA).

All mice are 6 weeks old at the beginning of the irradiation period. Animal Facilit
It is housed and maintained under standard conditions (23 ± 10 C, 42 ± 6% relative humidity, 12 hour light / dark cycle) in y of IBS. The lights are automatically turned on every day at 7am,
The lights are turned off every day at 7pm. Mice are Purina chow diet (24% protein, 4
% Fat, and 4.5% fiber), and is appropriately watered.
For irradiation, the mouse is placed in a plastic cage and can move freely in the cage between irradiations.

Prior to treatment, animals are acclimated to the environment for 1 week and randomly divided into 5 groups as follows. Group I: control, non-irradiated, untreated (n = 5), Group II: UV-unirradiated animals (n = 5) treated with topical ointment preparation containing FRST / 1, Group III : UV-irradiated animals treated with a topical ointment preparation without FRST / 1 (n = 5), Group IV: animals receiving a topical application of a cream containing FRST / 1 between UV irradiation (n = 5). = 5), Group V: animals that are untreated and exposed to UV light (n = 5) All animals are weighed for health assessment on the day of treatment and every second day thereafter. 2. Topical application of one type of cream, known to be non-toxic to animals and humans. This cream is with or without FRST / 1.
The extract is present at a 1: 10,000 dilution (starting with the lyophilized thylakoid fraction). One gram of cream is dripped in advance on the backs of the animals in three groups (and during and after UV irradiation). Of these, one group of 5 animals received the cream and no irradiation. One group is FR
Received cream containing no ST / 1 and exposed to UV B radiation, one group was FR
Receive ST / 1 cream and UV B. The group (n = 5) irradiated with the ultraviolet ray B is exposed to the sun lamp once for 10 minutes. One group (n = 5) is exposed to UV sun lamps without creaming. 3. A single 10 minute UV B irradiation is performed with a sun lamp placed 60 cm from the animal's back. Two groups of 5 animals are tested for cream protection (with and without FRST) and a single group of 5 animals without protection is exposed to a single dose of UVB. 4. After treatment, all animals are kept in cages and photographed from the top of the cages, without manipulation, on the day of UV irradiation and 3 to 4 days later. 5.1 weeks later, animals are sacrificed and their medicated skin strips excised for further investigation.

A Westinghouse FS40 solar lamp, IL-1400 radiometer, and UV B photometer are used. The irradiation spectrum of an ultraviolet lamp is 280-400 nm, of which 80
% Is the ultraviolet B region and 20% is the ultraviolet A region. The peak intensity of the light source is 297n
m. The fluence at 60 cm from the dosing surface of mice is 0.48 to 0.50
It is mJ / cm2 / s. Mice are placed in open cages as described above.

Negative control mice (Groups I, II) are treated in a similar fashion, but the UV lamp is not illuminated. Group II receives topical ointment without FRST / 1.

Mice in groups III, IV and V total 200 mJ UV / cm2 (
Acute exposure) for 10 minutes. In Group IV, animals receive topical application of the FRST / 1 preparation immediately before, during and after UV exposure. This method may affect UV irradiation conditions optically (Gonales and Pathak 1996) 290-3.
It is adjusted so as to take into account a slight difference in ultraviolet absorption characteristics (difference in optical density) in the range of 20 nm.

Mice are weighed and maintained every second day for 1 week before and 1 week after UV irradiation. At the end of the experiment, the mice were killed and the following parameters compared in all groups.

At the end of the experiment, the mice were killed and the following parameters are compared in all groups. 1. Weight 2. Observation result of epidermis and photograph 3. After killing the mice, the medicated skin was surgically excised and used for further analysis. 4. Comparison of cytokeratin expression patterns by quantitative Western blotting.

Solar irradiation is a major environmental factor affecting the structure and function of human skin.
Long-term UV damage to the skin as a result of accumulated UV radiation damage often leads to photoaging and skin cancer in individuals with fair skin. Studies, including photobiological studies of UV irradiation, show that the ultraviolet B (UVB) component (290-320 nm) is particularly erythematous, carcinogenic, and DNA, RNA, proteins (including enzymes), cell membranes and We show that direct damage to other cellular organelles causes pre-existing photoaging of the skin. Tedesco et al. (1997).

Kligman and Kligman (1993) made a clear distinction between chronological aging and ray aging. Light aging is used to describe the clinical and histological damage caused by chronic skin exposure to sunlight or solar pseudoultraviolet light. Histologically, these changes are manifested in the notable changes in elasticity, glycosaminoglycans, and disordered collagen coupled with an increase in mast and inflammatory cells (Kligman and G
ebre 1991; Kaaresn and Poulsen 1995; Poulsen et al., 1984). This phenomenon of photo-aging is recognized as clinically irreversible, even though recent therapeutic solutions help minimize the development of endogenous and photo-aging changes (Gilchrest 1996; Kang
And Voorhees 1998). It has been reported that the use of sunscreen agents on regular bases helps prevent light damage to connective tissue (Snyder and May 1975; Klig).
man and Kligman 1982; Bissett et al., 1991). The use of both sunscreens and antioxidants (eg green tea, vitamin C, vitamin E) has an inhibitory effect on UV-B-induced acute skin damage that contributes to both photoaging and photocarcinogenesis (Synder And May, 1
975; Bissett et al., 1991 and Huang et al., 1997).

The photoprotective or antioxidant effects of FRST / 1 against acute UV exposure have been investigated in a hairless, albino mouse model.

The hairless albino mouse (SKH-1) has been recognized as a useful and relevant experimental model to study and understand the effects of UV irradiation and photoaging in humans (Kligma).
n et al., 1989; Bissett et al., 1987; Chatterjee et al., 1990; Kligman and Gebre, 1991).
The visible and microscopically distinguishable epidermal and dermal responses to UV B irradiation and the absence of hair make the skin of hairless mice particularly useful for studying and assessing the damaging effects of UV irradiation. This mouse model has also been used both to study and test immunological changes and carcinogenesis induced by UVB irradiation locally and systemically on the skin (Fisher et al., 1989; Ho et al. , 1992; Reeve et al., 1991).

Results Precisely after irradiation, all untreated irradiated mice showed signs of skin inflammation and pruritus. Otherwise, they did not gain weight but were healthy and active. Similarly, when pretreated with FRST / 1, post-treatment did not show any symptoms of inflammation or reddening in irradiated mice, and increased weight in 80% of the cases.

Cutaneous cytokeratin analysis Cytokeratins 1 and 10 are symbols of mature skin. The loss of these keratins is a sign of epidermal regeneration after injury.

Cytokeratins 5 and 8 represent the upper and basal lamina and are assumed to be expressed in actively proliferating epidermis.

[0212]   The presence of these cytokeratins was examined with specific antibodies.

Mouse skin extracts were extracted individually and mouse skin was analyzed individually for each group. The same amount of the total extracted cytoplasmic protein was added to the wells. The amount that allowed comparison between animals and between treatments was 35 μg.

Conclusions FRST / 1 protected mice showed a pattern similar to that of control untreated and unirradiated, whereas all other treatments showed a dramatic reduction in high molecular weight keratin.
In particular, cytokeratin 10 continues to be expressed very well in the skin of FRST / 1 mice, which is a good sign of protective effect. The extract was topically active at very low doses. The dose of this extract is not limited by toxicity and the dose can be increased as desired.

While the present invention has been described above in the form of its preferred embodiments, it can be modified without departing from the spirit and nature of the subject invention as defined in the appended claims.

[Table 6]

[0216]

[References]

[Brief description of drawings]

FIG. 1 shows the relative antioxidant activity of extracts of the invention as a function of pH of external extracts.

FIG. 2 shows the relative activity of the extract of the present invention as a function of the concentration of sorbitol contained in the extract.

FIG. 3 shows the relative activity of the extract of the present invention as a function of time and the concentration of sugar contained in the extract. Each sample was kept at -20 ° C.

FIG. 4 shows the relative activity of the extracts of the invention as a function of the nature of the sugars in the extract.

FIG. 5 shows the relative activity of extracts of the invention as a function of various salts used for extraction.

FIG. 6 shows the relative activity of extracts of the invention (FRTS destabilized) as a function of time and temperature.

[Figure 7]   It is a figure which shows selection of the centrifugation conditions for optimization.

FIG. 8 shows the relative activity of the extracts of the invention as a function of the proportion of propylene glycol (propane-1,2-diol) contained in the resuspended solution.

FIG. 9 is a diagram showing an oxidation curve. Curve A shows lipid oxidation in the absence of antioxidants, curve B shows lipid oxidation in the presence of antioxidants that "permit" some lipid oxidation, and curve C shows that of vitamin E and the like. 3 shows lipid oxidation in the presence of effective radical chain-breaking antioxidants.

FIG. 10 is a diagram showing the oxidation rate of lipid PLPC, in which B represents the lipid without the extract of the present invention, D represents the lipid in the presence of Trolox, and L was prepared according to the present invention. Represents lipids in the presence of active extract, and T represents lipids in the presence of lipids treated with inactive extract.

FIG. 11A shows the protection of IMR-32 cells with the extract of the present invention (dilution 1: 1000) against injury caused by 25 μM of TBPH.

FIG. 11B shows the protection of IMR-32 cells with the extract of the present invention (dilution 1: 1000) against injury caused by 50 μM of TBPH.

FIG. 12A shows protection of IMR-32 cells treated with TBPH with a dilution of the extract of the invention of 1: 1000.

FIG. 12B shows protection of IMR-32 cells after TBPH treatment with a dilution of the extract of the invention of 1: 10000.

FIG. 13: Behavior of the hippocampus on hyperoxia for 120 seconds and inclusion of an extract according to the invention,
FIG. 5 shows recovery in the presence of or without (control) solution.

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] February 25, 2002 (2002.2.25)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0006

[Correction method] Change

[Contents of correction]

Photosynthetic pigments are extremely diverse. There are many different types of (bacterio) chlorophylls, carotenoids and phycobilins that differ from each other in exact chemical structure. Dyes are generally associated with proteins that bring the dye molecules into proper orientation and alignment with each other. Light energy is absorbed by the individual dyes, but is not immediately used by these dyes for energy conversion. Instead, light energy is passed to chlorophyll in a special protein environment, where the actual energy conversion phenomenon takes place. That is, light energy is used to transfer electrons to adjacent dyes. The dye and protein that are involved in this actual initial electron transfer phenomenon are called reaction centers. A large number of dye molecules (100 to 50)
00) "collects" light, traps photons and transfers light energy to the same reaction center. The purpose is to maintain a high electron transfer rate in the reaction center, even at low light intensities. The name P680 has been assigned to the chlorophyll dye of the reaction center PSII, because the chlorophyll pair having its composition mainly absorbs light having a wavelength of 680 nm.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0034

[Correction method] Change

[Contents of correction]

Thylakoids with optimized configuration and carotenoid ratio appear to retain sufficient activity, especially against ROS. Such antioxidants may be useful in reducing the expression of diseases and disorders involving ROS production. Such diseases and disorders may have etiologies associated with inflammation, cancer and radiation exposure. Such diseases and disorders include skin such as burns, sunburn, psoriasis, dermatitis; trauma, stroke,
Brains such as Parkinson's disease, neurotoxin, dementia, Alzheimer's disease; joints such as rheumatoid arthritis and arthritis; gastrointestinal tract such as diabetes, pancreatitis, endotoxin liver injury, intestinal ischemia, cataract development, retinopathy, degenerative retinal disorder, etc. Eyes; vascular vessels such as atherosclerosis and vasculitis; red blood cells such as Fanconi anemia and malaria; heart such as coronary artery thrombosis; lungs such as asthma and COPD;
Kidneys such as transplantation and glomerulonephritis; inflammation, cancer, ischemia-reperfusion, drug poisoning, iron overdose, malnutrition, alcohol poisoning, radiation, aging, amyloid disease, toxic shock, etc. Including those that affect. Literature on the involvement of ROS in some diseases is:

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0042

[Correction method] Change

[Contents of correction]

Biofilter / Bioreactor: The photosynthetic device in a plant can not only capture photons, but also capture and accumulate molecules having an affinity for each component thereof, and thus the extract of the present invention. Is imagined to have the same ability as the plant itself. Moreover, it is believed that the extract of the present invention acts as a bioreactor since it can process some of the capture molecules. Captured easily molecule, for example, herbicides, insecticides, fungicides, urea, ions and heavy metals, as well as _{O 3, CO, H 2 S} , NO, CO 2, gas such as O _2. The biofilter of the present invention is versatile and appears to be resistant to temperature changes.

[Procedure amendment 4]

[Document name to be amended] Statement

[Name of item to be corrected] 0057

[Correction method] Change

[Contents of correction]

The present invention is a method of obtaining an extract obtained from a photosynthetic organism containing thylakoids, comprising the steps of: a) providing a suspension of biocomponents containing thylakoids, and b) from 1 to 1.3 centipoise. In a medium having a viscosity of between 2 and a pH higher than 2 and lower than 10 while destroying its biogenic constituents while maintaining the thylakoid intact, the medium having a volume calculated according to ((Volume of medium + water content of plant constituents) / dry weight of plant constituents)> 10, thereby obtaining a first extract consisting essentially of thylakoids, cell debris / membrane and liquid phase, said A method is provided that comprises the step wherein the thylakoid comprises a complete photosynthetic pigment.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0209

[Correction method] Change

[Contents of correction]

Results Precisely after irradiation, all untreated irradiated mice showed signs of skin inflammation and pruritus. Otherwise, they did not gain weight but were healthy and active. Similarly, when pretreated with FRST / 1, post-treatment did not show any symptoms of inflammation or reddening in irradiated mice, and increased weight in 80% of the cases. Therefore, topical compositions, either sunscreen lotions, creams, ointments, oils, gels or sprays, are an object of the present invention.

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] April 15, 2002 (2002.4.15)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 1/18 A61P 1/18 3/10 3/10 7/00 7/00 7/06 7/06 9 / 00 9/00 9/10 9/10 11/00 11/00 11/06 11/06 13/12 13/12 17/00 17/00 17/02 17/02 17/06 17/06 19/02 19/02 25/00 25/00 25/16 25/16 25/28 25/28 25/30 25/30 25/32 25/32 27/02 27/02 27/12 27/12 29/00 29 / 00 101 101 31/04 31/04 33/06 33/06 35/00 35/00 37/06 37/06 39/00 39/00 39/06 39/06 43/00 105 43/00 105 C09B 61 / 00 C09B 61/00 B C09K 3/00 104 C09K 3/00 104Z 15/34 15/34 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR , IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, I, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW) , EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA , CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO , RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW F term (reference) 4C083 AA111 BB46 CC19 DD08 DD22 DD23 DD30 DD31 DD41 EE17 4C088 AB12 BA08 CA03 CA23 MA07 MA13 MA16 MA17 MA22 MA28 MA63 ZA01 ZA15 ZA16 ZA33 ZA36 ZA33 ZA36 ZA36 ZA33 ZA36 ZA36 ZA36 ZA36 ZA36 ZA66 ZA81 ZA89 ZA96 ZB11 ZB15 ZB21 ZB26 ZB35 ZC35 ZC37 ZC39 4H025 BA01 (54) [Invention] Method for obtaining thylakoids from photosynthetic organisms, plant fractions obtained from the method, pure thylakoids, and ROS scavengers, light protection To use thylakoids as agents, biosensors, biofilters and bioreactors

Claims (47)

[Claims] 1. A method for obtaining an extract from a photosynthetic organism, comprising the steps of a) providing a suspension of biocomponents comprising thylakoids, and b) from a viscosity comprised between 1 and 1.3 and 2. The step of destroying biological components while recovering thylakoids in a medium having a pH higher than 10 and adding a volume of the medium calculated according to the following formula: ((volume of medium + containing plant components) Water amount) / plant component dry weight)> 10, whereby a first extract consisting essentially of thylakoids, cell debris / membrane and liquid phase is obtained, said thylakoids comprising an organized photosynthetic pigment. How to include. 2. The equation is ((Volume of medium + water content of plant components) / dry weight of plant components) = 25-150 The method of claim 1, wherein 3. The method according to claim 1 or 2, wherein the pH is comprised between 5 and 8. 4. The method according to claim 1, wherein the pH is comprised between 6 and 7.5. 5. The method according to any one of claims 1 to 4, wherein the organism is a plant. 6. The method according to claim 5, wherein the suspension of step a) is obtained by mechanically dispersing the plant constituents or tissues in the medium. 7. Prior to step a), a step of subjecting the plant to conditioning parameters selected from light, osmotic pressure, heat, cold, freezing, drying, hormones, chemical and biological inducers is carried out. The method according to claim 5 or 6. 8. The method according to claim 1, wherein before step a) there is a step of conditioning said plant with a light environment having a wavelength comprised between about 500 and 600 nm and step b) is carried out under the same light environment. The method according to any one of 4 above. 9. The method according to claim 5, wherein before step a) there is a step of conditioning said plant with a light environment having a wavelength comprised between about 500 and 600 nm and step b) is carried out under the same light environment. 7. The method according to any one of 7. 10. The method according to any one of claims 1 to 9, wherein the viscosity is achieved in part by adding sugar in the medium. 11. The viscosity is partly due to the presence of saccharides in the medium at a concentration of about 0.2 to 1.5 M or sugars that achieve a viscosity equivalent to 0.2 to 1.5 M sorbitol. The method according to any one of claims 1 to 10, implemented in. 12. The viscosity is partly due to the presence of saccharides in the medium at a concentration of about 0.2 to 0.4 M or sugars that achieve a viscosity equivalent to 0.2 to 0.4 M sorbitol. The method according to any one of claims 1 to 10, implemented in. 13. The medium according to claim 1, wherein the medium has the following composition: Tris or acetate or ascorbate buffer (20 mM) having a pH of 7.5, or sorbitol or sucrose or fructose 350 mM. The method described in the section. 14. A further step c), namely by separating the thylakoids, cell debris / membrane and liquid phase from each other, essentially consisting of separated thylakoids, cell debris / membrane and liquid phase, respectively. 14. A method according to any one of claims 1 to 13 including the step of forming second, third and fourth extracts according to claim 1. 15. The method according to claim 14, wherein the separating step is carried out based on the difference of the sedimentation coefficients of thylakoid, cell debris and membrane, and liquid phase. 16. The separation step comprises centrifuging the first extract in a tube having a filter at the top of the tube, the filter comprising debris and cell debris while the thylakoid and liquid phases pass through the filter. 16. The method of claim 15, wherein the membrane has porosity deposited thereon and the thylakoid comprises forming a pellet at the bottom of the tube. 17. The photosynthetic dye is inactivated by a further step d), ie by erasing any electron donor from said first, second and third extracts,
17. A method according to any one of claims 14 to 16 including the step of stabilizing. 18. The method of claim 17, wherein the electron donor is water. 19. The method according to claim 18, wherein water is removed under vacuum freeze-drying. 20. The method according to claim 18, wherein after step c) the water is removed by exchanging the water with an amphoteric solvent or a surfactant. 21. The method of claim 18, wherein the water is removed by exchanging the water with propylene glycol. 22. A botanical extract which contains substantially pure thylakoids in its intact state. 23. The method according to claim 22, which can be further activated when an electron donor is added.
The plant extract according to. 24. An extract produced by the method according to any one of claims 1 to 16. 25. An extract which is essentially a first, second or third extract obtained from the method according to any one of claims 17 to 24. 26. An extract which is essentially a second extract obtained from the method according to any one of claims 17 to 24. 27. The extract according to claim 26, which is in a dry state. 28. The extract according to claim 26, which is partially or completely solubilized. 29. The extract according to claim 26, which is a suspension. 30. The extract according to any one of claims 24 to 29, which is activated in the presence of an electron donor. 31. The extract according to claim 23 or 30, wherein the electron donor is water. 32. A method according to claims 25 to 3 as a scavenger of reactive oxygen species.
Use of the extract according to any one of 1. 33. The use according to claim 32, which reduces the development of diseases or disorders involving the formation of reactive oxygen species. 34. The use according to claim 33, wherein the disease or disorder has an etiology associated with inflammation, cancer or radiation exposure. 35. The disease or disorder affects the skin and is selected from burns, sunburns, psoriasis and dermatitis; affects the brain and from trauma, stroke, Parkinson's disease, neurotoxins, dementia and Alzheimer's disease. Selected; affecting joints and selected from rheumatoid arthritis and arthropathy; affecting the gastrointestinal tract, and diabetes, pancreatitis,
Endotoxins selected from liver injury and intestinal ischemia; affecting the eye and selected from cataractogenesis, retinopathy and degenerative retinopathy; affecting vasculature and selected from atherosclerosis and vasculitis; Affects red blood cells and is selected from Fanconi anemia and malaria; affects heart and is selected from coronary thrombosis and ischemia;
Affecting lungs and selected from asthma and COPD; affecting kidneys and selected from transplantation and glomerulonephritis; affecting multiple organs and inflammation, cancer, ischemia-reperfusion,
34. Use according to claim 33, selected from drug poisoning, iron overdose, malnutrition, alcoholism, radiation, aging, amyloid disease and toxic shock. 36. The use according to claim 33, wherein the disease or disorder affects the skin or mucous membranes and its use is topical. 37. The use according to claim 34, wherein the radiation falls in the ultraviolet spectrum. 38. A filter according to claim 26 or 28 as a filter for radiation.
Use of the second or third extract as described in 1. 39. The use according to claim 38, wherein the radiation enters the ultraviolet region. 40. Use of the extract according to any one of claims 22 to 31 for trapping photons. 41. Use of the second and third extracts according to any one of claims 25 to 31 for detecting or capturing molecules which have an affinity for thylakoids or which interfere with thylakoid activity. . 42. The molecule is a triazine, atrazine type, diuron type herbicide, quinone, chlorpromazine, urea, formaldehyde, alkylaminocyanoacrylate, trypsin, cyanoacrylate, tris, adenine derivative,
Disulfiran, acetyl CoA carboxylase, digitonin, heavy metals, Cu, Zn
, Cd, Pb, Hg, SO ₂ , NO ₂ , NH ₂ OH, CO ₂ , CO, O ₃ , O ₂ , H ₂ S, calcium antagonist,
Selected from ^- sulfates, sulfites, bisulfites, nitrite, acetate (salt), lactate (salt), ^{_{anionic, NO 3 -, HCO 3 -}} , HCO 2 -, F -, NO 2 - and HSO ₃ 42. Use according to claim 41. 43. A substance composition comprising the extract according to any one of claims 25 to 31. 44. The composition of claim 43 which is a topical composition. 45. The composition of claim 44 which is a sunscreen lotion, cream, ointment, oil, gel or spray. 46. A device or instrument comprising the extract according to any one of claims 25 to 31. 47. The device or instrument of claim 46 which is a bioreactor, biofilter or biosensor.